Compositions and methods for modulating lymphocyte activity

ABSTRACT

The invention derives from the identification of HVEM as the native ligand for BTLA. The invention provides compositions and methods for modulating BTLA-HVEM interactions and BTLA and HVEM activity, which are useful for modulating immune responses. Agonists and antagonists of the BTLA-HVEM interaction are provided, and methods of treating a variety of conditions through the modulation of immune responses are provided.

STATEMENT OF RELATEDNESS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/628,474, filed Nov. 15, 2004, the disclosure of which isincorporated by reference.

FIELD

The present invention relates to mechanisms of immunomodulation, as wellas to compositions and methods useful for modulating an immune responsefor therapeutic purposes. The invention relates to the treatment ofcancer, infection, autoimmune disease, inflammation, and allergy byimmunomodulatory means. The invention further relates to the treatmentof transplant recipients, and to the reduction of graft versus hostreactions in patients by immunomodulatory means.

BACKGROUND

Positive and negative costimulatory signals play critical roles in themodulation of T cell activity. Positive costimulation, in addition to Tcell receptor (TCR) engagement, is required for optimal activation ofnaïve T cells, whereas negative costimulation is believed to be requiredfor the acquisition of immunologic tolerance to self, as well as thetermination of effector T cell functions. Upon interaction with B7.1 orB7.2 on the surface of antigen-presenting cells (APC), CD28, theprototypic T cell costimulatory molecule, emits signals that promote Tcell proliferation and differentiation in response to TCR engagement,while the CD28 homologue cytotoxic T lymphocyte antigen-4 (CTLA-4) emitssignals that inhibit T cell proliferation and effector functions(Chambers et al., Ann. Rev. Immunol., 19:565-594, 2001; Egen et al.,Nature Immunol., 3:611-618, 2002).

Agents capable of modulating positive and negative costimulatory signalsare highly desirable for use in the modulation of adaptive immuneresponses. Many autoimmune disorders are known to involve autoreactive Tcells and autoantibodies. Agents that are capable of inhibiting theactivation of lymphocytes that are specific for self antigens aredesirable. Similarly, under certain conditions it is desirable toinhibit normal immune responses to antigen. For example, the suppressionof normal immune responses in a patient receiving a transplant isdesirable, and agents that exhibit such immunosuppressive activity arehighly desirable.

Conversely, many cancer immunotherapies, such as adoptive immunotherapy,expand tumor-specific T cell populations and direct them to attack andkill tumor cells (Dudley et al., Science 298:850-854, 2002; Pardoll,Nature Biotech., 20:1207-1208, 2002; Egen et al., Nature Immunol.,3:611-618, 2002). Agents capable of augmenting tumor attack are highlydesirable.

In addition, immune responses to many different antigens (e.g.,microbial antigens or tumor antigens), while detectable, are frequentlyof insufficient magnitude to afford protection against a diseaseprocess. Agents capable of promoting and/or prolonging the activation(delaying termination) of lymphocytes that are specific for suchantigens are highly desirable.

Costimulatory signals, particularly positive costimulatory signals, alsoplay a role in the modulation of B cell activity. For example, B cellactivation and the survival of germinal center B cells require Tcell-derived signals in addition to stimulation by antigen. CD40 ligandpresent on the surface of helper T cells interacts with CD40 on thesurface of B cells and provides such a positive costimulatory signal toB cells.

Recently, a negative costimulatory receptor analogous to CTLA-4 wasidentified on B cells and T cells (Watanabe et al., Nat. Immunol.,4:670-679, 2003; U.S. patent application Ser. No. 10/600,997, filed 20Jun. 2003; both of which are expressly incorporated herein in theirentirety by reference). B and T lymphocyte attenuator (BTLA) is animmunoglobulin domain-containing glycoprotein with a Grb2 binding site,an immunoreceptor tyrosine-based inhibitory motif (ITIM), and animmunoreceptor tyrosine-based switch motif (ITSM). Partial BTLAsequences were disclosed previously (WO 99/40100 and WO 02/07294) thoughthe complete sequence, distribution, and function of BTLA was notreported. Additionally, the partial BTLA sequences disclosed wereasserted to correspond to secreted proteins rather than a functionalreceptor on the surface of lymphocytes.

BTLA acts a negative regulator of both B and T lymphocyte activity(Watanabe et al., Nat. Immunol., 4:670-679, 2003). Crosslinking BTLAwith antigen receptors induces its tyrosine phosphorylation andassociation with the Src homology domain 2 (SH2)-containing proteintyrosine phosphatases SHP-1 and SHP-2, and attenuates production ofinterleukin 2 (IL-2). BTLA-deficient T cells show increasedproliferation, and BTLA-deficient mice have increased specific antibodyresponses and enhanced sensitivity to experimental autoimmuneencephalomyelitis.

Based on indirect evidence, the ligand for BTLA was previously assertedto be B7x (Watanabe et al., supra). However, as disclosed herein, B7xdoes not bind to BTLA. The identification of BTLA's cognate ligand thusremains highly desirable for an understanding of BTLA function, and fordiagnostic and therapeutic purposes.

Herpes virus entry mediator (“HVEM”), a member of the TNF/NGF receptorfamily, is another positive costimulatory receptor that additionallymediates the entry of herpes simplex virus (HSV) into cells (Montgomeryet al., Cell. 1996 Nov. 1; 87(3):427-36). Anti-HVEM antibodies and asoluble hybrid protein containing the HVEM ectodomain have been shown toinhibit such HVEM-dependent viral entry. HSV-1 glycoprotein D (gD), astructural component of the HSV envelope, binds to HVEM to facilitateviral entry (Whitbeck et al., J. Virol. 1997 August; 71(8):6083-93).HVEM binds two cellular ligands, secreted lymphotoxin alpha and LIGHT(Mauri et al., Immunity. 1998 January; 8(1):21-30). HSV-1 gD inhibitsthe interaction of HVEM with LIGHT. Additionally, targeted disruption ofLIGHT causes immunomodulatory defects (Scheu et al., J. Exp. Med.,195:1613-1624, 2002). Additionally, a phage-derived peptide BP-2reportedly binds to HVEM and can compete with HSV-1 gD (Carfi et al.,Mol. Cell. 8:169-179, 2001; Sarrias et al., Mol. Immunol., 37:665-673,2000).

SUMMARY OF THE INVENTION

The present disclosure establishes that Herpes virus entry mediator(HVEM) is the cognate ligand of BTLA. HVEM belongs to the TNF receptorfamily of proteins and is itself a costimulatory receptor expressed onnaive T cells. HVEM is also expressed to a lesser extent on dendriticcells, resting B cells, and macrophages. HVEM has four extracellularcysteine-rich domains (CRDs) and interacts with two known TNF familymembers, LIGHT and lymphotoxin alpha (LTα), through CRD2 and CRD3. Forfurther discussion of HVEM, see for example Granger et al., CytokineGrowth Factor Rev., 14:289-96, 2003; and Croft, Nat. Rev. Immunol.,3:609-620, 2003. As disclosed herein, HVEM directly binds to BTLA andstimulates BTLA activity. As further disclosed herein, HVEM binding toBTLA can reduce the activation of BTLA expressing lymphocytes, as wellas decrease the effector activity of BTLA expressing lymphocytes.

The present disclosure also establishes that B7x does not directly bindto BTLA and does not directly modulate BTLA activity. B7x is expressedin a wide variety of normal and cancer cells, and was previouslyreported to be a ligand for BTLA based on indirect evidence. It waspostulated that the interaction of B7x with BTLA inhibited both B and Tcell responses, and was a means by which B7x-expressing tumor tissueinhibited the activity of tumor-specific T cells. It was further positedthat B7x expressed on non-tumor non-lymphoid tissue served to maintainimmunological tolerance to self antigens.

Stemming from the discovery of the HVEM-BTLA interaction, in one aspect,the present invention provides BTLA antibodies, sometimes referred toherein as BTLA blocking antibodies. A BTLA antibody of the invention iscapable of specifically binding to a BTLA protein and is capable ofreducing the binding of the BTLA protein to an HVEM protein. Especiallypreferred are BTLA antibodies that specifically bind to a region of theBTLA Ig domain, which region binds to the HVEM CRD1 domain. Such a BTLAantibody is capable of binding to a fragment of the BTLA Ig domain,which fragment is capable of binding to an HVEM CRD1 domain.

In one embodiment, a BTLA antibody is capable of binding to a mouse BTLAIg domain.

In one embodiment, a BTLA antibody is capable of binding to a mouse BTLAIg domain in a human BTLA tetramer.

In one embodiment, a BTLA antibody is capable of binding to a human BTLAIg domain.

In one embodiment, a BTLA antibody is capable of binding to a human BTLAIg domain in a human BTLA tetramer.

In one embodiment, a BTLA antibody is capable of binding toward the DEBAface of the Ig fold of BTLA. The phrase “DEBA face” refers to theregions of the BTLA molecule composed of the beta strands labelled “D”,“E”, “B”, and “A” strands. See, for example, structure of BTLAectodomain deposited at NCBI by C. A. Nelson, D. H. Fremont, MidwestCenter For Structural & Genomics (Mcsg), 26 Aug. 4. See also Compaan etal., J Biol. Chem. 2005 Sep. 16, Epub manuscript M507629200.

In one embodiment, a BTLA antibody is capable of binding an epitope ofBTLA that is capable of binding to an antibody selected from the groupconsisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’.

In one embodiment, a BTLA antibody is capable of competing with anantibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’,‘6H6’, ‘8F4’, and ‘3F9.D12’ for binding to BTLA.

In one embodiment, a BTLA antibody is capable of binding to an epitopeof BTLA that is homologous to an epitope capable of binding an antibodyselected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’,and ‘3F9.D12’.

In one embodiment, a BTLA antibody is capable of binding to an epitopecomprising one or more residues selected from the group consisting ofR55, Q63, Q 102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA antibody is capable of binding to an epitopecomprising one or more residues selected from the group consisting ofthe residues in a BTLA protein corresponding to the residues V42, Q43,L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA antibody is capable of binding to an epitopecomprising one or more residues selected from the group consisting ofthe residues in human BTLA corresponding to the residues V42, Q43, L44,R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA antibody is capable of binding to an epitopecomprising one or more residues selected from the group consisting ofV36, Q37, L38, L49, E57, C79, K93, and S96 in the human BTLA sequenceset forth at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA antibody is capable of binding to an epitopecomprising one or more residues in a human BTLA corresponding toresidues from the group consisting of V36, Q37, L38, L49, E57, C79, K93,and S96 in the human BTLA sequence set forth at Genbank accession no.AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA antibody is capable of binding to apolypeptide having at least about 80%, more preferably 85%, morepreferably 90%, more preferably 95% identity to the amino acid sequenceset forth by residues 37-47, 39-49, 41-49, 50-60, 58-68, 80-90, 97-107,50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137, 55-102, 50-107, and41-137 of murine BI/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA antibody is capable of binding to apolypeptide selected from the group consisting of the amino acidsequences set forth by residues 37-47, 39-49, 41-49, 50-60, 58-68,80-90, 97-107, 50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137,55-102, 50-107, and 41-137 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA antibody is capable of binding to apolypeptide having at least about 80%, more preferably 85%, morepreferably 90%, more preferably 95% identity to the amino acid sequenceset forth by residues 31-41, 32-42, 35-43, 44-54, 52-62, 74-84, 88-98,44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 49-93, 44-98, 35-98,and 35-128 of the human BTLA isoform found at Genbank accession no.AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA antibody is capable of binding to apolypeptide selected from the group consisting of the amino acidsequences set forth by residues 31-41, 32-42, 35-43, 44-54, 52-62,74-84, 88-98, 44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 49-93,44-98, 35-98, and 35-128 of the human BTLA isoform found at Genbankaccession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA antibody is selected from the group consistingof ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’.

In one embodiment, a BTLA antibody is capable of competing with CMVUL144 for binding to BTLA.

In one embodiment, the invention provides BTLA antibodies which aremonoclonal antibodies.

In one embodiment, the invention provides BTLA antibodies which arehuman antibodies.

In one aspect, the invention provides a hybridoma that produces a BTLAantibody disclosed herein.

In one aspect, the invention provides BTLA antibodies that are capableof modulating BTLA activity.

In one embodiment, the invention provides BTLA antibodies that areantagonistic BTLA antibodies, which are capable of reducing BTLAactivity. Such antibodies are capable of reducing the activation of BTLAby HVEM. Preferably, such antagonistic BTLA antibodies are also capableof reducing the activation of BTLA by another ligand which binds to theHVEM binding region of BTLA, such as UL144. The UL144 open reading framein human cytomegalovirus (CMV) encodes a homologue of the herpesvirusentry mediator, HVEM, a member of the tumor necrosis factor receptorsuperfamily (Lurain et al., J. Virol. 1999 December; 73(12):10040-50).

In another embodiment, the invention provides BTLA antibodies that areagonistic BTLA antibodies, which are capable of increasing BTLAactivity. Such antibodies are capable of increasing BTLA activity in acell having BTLA on its surface.

In one aspect, the invention provides HVEM antibodies, sometimesreferred to herein as HVEM blocking antibodies. An HVEM antibodyspecifically binds to an HVEM protein and is capable of reducing thebinding of the HVEM protein to a BTLA protein. Especially preferred areHVEM antibodies that specifically bind to a region of the HVEM CRD1domain that binds to the BTLA Ig domain. Such an HVEM antibody iscapable of binding to a fragment of the HVEM CRD1 domain, which fragmentis capable of binding to a BTLA Ig domain. Preferred HVEM antibodies donot bind to the HVEM CRD2 or HVEM CRD3 domains, though antibodiesbinding to the CRD2 and/or CRD3 domains in addition to the CRD1 domainmay be used in the methods herein.

In one embodiment, the invention provides HVEM antibodies which aremonoclonal antibodies.

In one aspect, the invention provides a hybridoma that produces a HVEMantibody disclosed herein.

In one embodiment, the invention provides HVEM antibodies which arehuman antibodies. In one aspect, the invention provides HVEM antibodiesthat are capable of modulating BTLA activity.

In a preferred embodiment, the invention provides HVEM antibodies thatare antagonistic HVEM antibodies, which are capable of reducing theability of HVEM to activate BTLA on the surface of a cell.

In another embodiment, the invention provides HVEM antibodies that areagonistic HVEM antibodies, which are capable of binding to HVEM andstimulating HVEM activity in a cell, thereby mimicking BTLA. HVEMactivity in this sense includes increased NF-kB activity and increasedAP-1 activity.

In one embodiment, the invention provides HVEM antibodies that do notinhibit the binding of HVEM to LIGHT or LTα.

In one embodiment, the invention provides HVEM antibodies thatadditionally reduce the binding of HSV-1 glycoprotein D to HVEM.

In one aspect, the invention provides BTLA-HVEM antagonists. A BTLA-HVEMantagonist may be any of a wide variety of bioactive agents capable ofreducing the activation of BTLA by HVEM. In a preferred embodiment, aBTLA-HVEM antagonist is capable of reducing the binding of an HVEM CRD1domain to a BTLA Ig domain. While many BTLA-HVEM antagonists are capableof binding to BTLA, such a BTLA-HVEM antagonist does not increase BTLAactivity in a cell expressing BTLA on its surface.

Preferred BTLA-HVEM antagonists are capable of reducing BTLA activity ina cell having BTLA on its surface. In a preferred embodiment, the cellis a lymphocyte, a T cell, a CD4⁺ T cell, a T_(H)1 cell, a CD8⁺ T cell,a B cell, a plasma cell, a macrophage, or an NK cell.

Suitable bioactive agents include BTLA antibodies and HVEM antibodies(e.g., monoclonal, polyclonal, single chain, and/or bispecificantibodies as well as Fab and F(ab)₂ fragments, variants and derivativesthereof). Suitable bioactive agents also include fragments and truncatedforms of BTLA and HVEM proteins, fusion proteins, and the like, forexample, soluble proteins and polypeptides comprising or consistingessentially of a BTLA Ig domain fragment capable of binding an HVEM CRD1domain; soluble proteins and polypeptides comprising or consistingessentially of an HVEM CRD1 domain or fragment thereof capable ofbinding a BTLA Ig domain; a BTLA Ig domain peptide, a CRD1 domainpeptide. Suitable bioactive agents also include small molecule chemicalcompositions.

In one embodiment, the invention provides a BTLA-HVEM antagonist capableof reducing the binding of a BTLA protein to an HVEM protein, whereinthe antagonist does not comprise an HVEM CRD2 domain, an HVEM CRD3domain, or both, and wherein the antagonist does not bind to an HVEMCRD2 domain or an HVEM CRD3 domain, with the proviso that the antagonistis not an HSV-1 glycoprotein D, a phage-derived peptide BP-2, or asoluble protein comprising a complete BTLA Ig domain capable of bindingsaid HVEM protein.

In the methods herein, glycoprotein D and phage-derived peptide BP-2, aswell as HVEM-binding fragments thereof, and fusion proteins comprisingthe same, may be used as BTLA-HVEM antagonists.

Preferred BTLA-HVEM antagonists are capable of binding to a BTLA Igdomain and are capable of reducing the binding of the BTLA Ig domain toan HVEM CRD1 domain. Especially preferred are BTLA-HVEM antagonistscapable of binding to a region of the BTLA Ig domain that binds to theHVEM CRD1 domain. Such a BTLA-HVEM antagonist is capable of binding to afragment of the BTLA Ig domain, which fragment is capable of binding toan HVEM CRD1 domain.

In one embodiment, a BTLA-HVEM antagonist binds an epitope of BTLA thatis capable of binding to an antibody selected from the group consistingof ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’.

In one embodiment, a BTLA-HVEM antagonist is capable of competing withan antibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’,‘6H6’, ‘8F4’, and ‘3F9.D12’ for binding to BTLA.

In one embodiment, a BTLA-HVEM antagonist binds to an epitope of BTLAthat is homologous to an epitope capable of binding an antibody selectedfrom the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and‘3F9.D12’.

In one embodiment, a BTLA-HVEM antagonist is capable of binding to anepitope comprising one or more residues selected from the groupconsisting of V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to anepitope comprising one or more residues selected from the groupconsisting of the residues in a BTLA protein corresponding to theresidues V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA(SEQ ID NO:1).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to anepitope comprising one or more residues selected from the groupconsisting of the residues in human BTLA corresponding to the residuesV42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ IDNO:1).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to anepitope comprising one or more residues selected from the groupconsisting of V36, Q37, L38, L49, E57, C79, K93, and S96 in the humanBTLA sequence set forth at Genbank accession no. AAP44003.1 (SEQ IDNO:2).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to anepitope comprising one or more residues of human BTLA corresponding toresidues from the group consisting of V36, Q37, L38, L49, E57, C79, K93,and S96 in the human BTLA sequence set forth at Genbank accession no.AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to apolypeptide having at least about 80%, more preferably 85%, morepreferably 90%, more preferably 95% identity to the amino acid sequenceset forth by residues 37-47, 39-49, 41-49, 50-60, 58-68, 80-90, 97-107,50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137, 55-102, 50-107, and41-137 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to apolypeptide selected from the group consisting of the amino acidsequences set forth by residues 37-47, 39-49, 41-49, 50-60, 58-68,80-90, 97-107, 50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137,55-102, 50-107, and 41-137 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to apolypeptide having at least about 80%, more preferably 85%, morepreferably 90%, more preferably 95% identity to the amino acid sequenceset forth by residues 31-41, 32-42, 35-43, 44-54, 52-62, 74-84, 88-98,44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 49-93, 44-98, 35-98,and 35-128 of the human BTLA isoform found at Genbank accession no.AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to apolypeptide selected from the group consisting of the amino acidsequences set forth by residues 31-41, 32-42, 35-43, 44-54, 52-62,74-84, 88-98, 44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 49-93,44-98, 35-98, and 35-128 of the human BTLA isoform found at Genbankaccession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM antagonist is capable of competing withCMV UL144 for binding to BTLA.

In one embodiment, a BTLA-HVEM antagonist is capable of competing withHSV-1 glycoprotein D for binding to HVEM.

In one embodiment, a BTLA-HVEM antagonist is a BTLA antibody.

In one embodiment, a BTLA-HVEM antagonist is an HVEM antibody.

In one aspect, the invention provides BTLA-HVEM antagonists thatcomprise a BTLA Ig domain fragment capable of binding an HVEM CRD1domain. In another aspect, the invention provides BTLA-HVEM antagoniststhat consist essentially of a BTLA Ig domain fragment capable of bindingan HVEM CRD1 domain.

Accordingly, in a preferred embodiment, the invention provides BTLA-HVEMantagonists that are BTLA fusion proteins which are capable of bindingto an HVEM CRD1 domain and reducing the binding of the HVEM CRD1 domainto a BTLA Ig domain. Preferred BTLA fusion proteins do not bind to theCRD2 or CRD3 domains of HVEM. Preferred BTLA fusion proteins can competewith an HVEM antibody disclosed herein for binding to an HVEM CRD1domain. Preferred BTLA fusion proteins do not comprise an entire BTLA Igdomain.

In another preferred embodiment, the invention provides BTLA-HVEMantagonists that are BTLA protein fragments which are capable of bindingto the CRD1 domain of HVEM and reducing the binding of the HVEM CRD1domain to a BTLA Ig domain. Preferred BTLA protein fragments do not bindto the CRD2 or CRD3 domains of HVEM. In a preferred embodiment, a BTLAprotein fragment consists essentially of a BTLA Ig domain fragment thatis capable of binding to an HVEM CRD1 domain. Preferred BTLA proteinfragments can compete with an HVEM antibody disclosed herein for bindingto an HVEM CRD1 domain.

In one aspect, the invention provides BTLA-HVEM antagonists thatcomprise an HVEM CRD1 domain or fragment thereof capable of binding to aBTLA Ig domain. In another aspect, the invention provides BTLA-HVEMantagonists that consist essentially of an HVEM CRD1 domain or fragmentthereof capable of binding to a BTLA Ig domain.

Accordingly, in a preferred embodiment, the invention provides BTLA-HVEMantagonists that are HVEM fusion proteins which are capable of bindingto a BTLA Ig domain and reducing the binding of the BTLA Ig domain to anHVEM CRD1 domain. Such HVEM fusion proteins lack an HVEM CRD2 and/orCRD3 domain. Preferred HVEM fusion proteins can compete with a BTLAantibody disclosed herein for binding to a BTLA Ig domain.

In another preferred embodiment, the invention provides BTLA-HVEMantagonists that are HVEM protein fragments which are capable of bindingto a BTLA Ig domain and reducing the binding of the BTLA Ig domain to anHVEM CRD1 domain. Such HVEM protein fragments lack an HVEM CRD2 and/orCRD3 domain. In a preferred embodiment, an HVEM protein fragmentconsists essentially of an HVEM CRD1 domain or fragment thereof which iscapable of binding to a BTLA Ig domain. Preferred HVEM protein fragmentscan compete with a BTLA antibody disclosed herein for binding to a BTLAIg domain.

In one embodiment, the invention provides fusion proteins that comprisean Fc region of an immunoglobulin.

In one embodiment, for use in the methods herein, HSV-1 glycoprotein Dmay be used as a BTLA-HVEM antagonist.

In one embodiment, a BTLA-HVEM antagonist is capable of reducingtyrosine phosphorylation on the intracellular domain of BTLA protein ina cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM antagonist is capable of reducingassociation of BTLA protein with SHP-2, PI3K, or Grb2 in a cell havingBTLA protein on its surface.

In one embodiment, a BTLA-HVEM antagonist is capable of increasingproliferation of a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM antagonist is capable of increasing IL-2production by a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM antagonist is capable of increasing orprolonging antibody production by a cell having said BTLA protein on itssurface.

In one embodiment, a BTLA-HVEM antagonist is capable of increasing orprolonging the cytotoxicity of a cell having said BTLA protein on itssurface.

In one aspect, the invention provides BTLA-HVEM agonists. A BTLA-HVEMagonist may be any of a wide variety of bioactive agents capable ofactivating BTLA and thereby mimicking the activity of HVEM.

Preferred BTLA-HVEM agonists are capable of increasing BTLA activity ina cell having BTLA on its surface. In a preferred embodiment, the cellis a lymphocyte, a T cell, a CD4⁺ T cell, a T_(H)1 cell, a CD8⁺ T cell,a B cell, a plasma cell, a macrophage, or an NK cell.

Suitable bioactive agents include BTLA antibodies (e.g., monoclonal,polyclonal, single chain, and/or bispecific antibodies as well as Faband F(ab)₂ fragments, variants and derivatives thereof). Suitablebioactive agents also include fragments and truncated forms of HVEMproteins, fusion proteins, and the like, such as soluble proteins andpolypeptides comprising or consisting essentially of an HVEM CRD1 domainor fragment thereof capable of binding to a BTLA Ig domain andincreasing BTLA activity, and lacking a CRD2 and/or CRD3 domain.Suitable bioactive agents also include small molecule chemicalcompositions.

In one embodiment, the invention provides a BTLA-HVEM agonist capable ofbinding to BTLA protein and increasing BTLA activity, wherein theagonist does not comprise an HVEM CRD2 domain, an HVEM CRD3 domain, orboth, with the proviso that the agonist is not a human CMV UL144protein.

In one embodiment, for use in the methods herein, CMV UL144,BTLA-binding fragments thereof, and fusion proteins comprising the same,may be used as a BTLA-HVEM agonist. Further regarding UL144, see Cheunget al., PNAS 102:13218-13223, 2005.

Preferred BTLA-HVEM agonists bind to a BTLA Ig domain and are capable ofreducing the binding of the BTLA Ig domain to an HVEM CRD1 domain, andmimicking the stimulation of BTLA by HVEM. Especially preferred areBTLA-HVEM agonists are capable of binding to a region of the BTLA Igdomain that binds to the HVEM CRD1 domain.

In one embodiment, a BTLA-HVEM agonist binds an epitope of BTLA that iscapable of binding to an antibody selected from the group consisting of‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’.

In one embodiment, a BTLA-HVEM agonist is capable of competing with anantibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’,‘6H6’, ‘8F4’, and ‘3F9.D12’ for binding to BTLA.

In one embodiment, a BTLA-HVEM agonist binds to an epitope of BTLA thatis homologous to an epitope capable of binding an antibody selected fromthe group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and‘3F9.D12’.

In one embodiment, a BTLA-HVEM agonist is capable of binding to anepitope comprising one or more residues selected from the groupconsisting of V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM agonist is capable of binding to anepitope comprising one or more residues selected from the groupconsisting of the residues in a BTLA protein corresponding to theresidues V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA(SEQ ID NO:1).

In one embodiment, a BTLA-HVEM agonist is capable of binding to anepitope comprising one or more residues selected from the groupconsisting of the residues in human BTLA corresponding to the residuesV42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ IDNO:1).

In one embodiment, a BTLA-HVEM agonist is capable of binding to anepitope comprising one or more residues selected from the groupconsisting of V36, Q37, L38, L49, E57, C79, K93, and S96 in the humanBTLA sequence set forth at Genbank accession no. AAP44003.1 (SEQ IDNO:2).

In one embodiment, a BTLA-HVEM agonist is capable of binding to anepitope comprising one or more residues in human BTLA corresponding toresidues from the group consisting of V36, Q37, L38, L49, E57, C79, K93,and S96 in the human BTLA sequence set forth at Genbank accession no.AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM agonist is capable of binding to apolypeptide having at least about 80%, more preferably 85%, morepreferably 90%, more preferably 95% identity to the amino acid sequenceset forth by residues 37-47, 39-49, 41-49, 50-60, 58-68, 80-90, 97-107,50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137, 55-102, 50-107, and41-137 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM agonist is capable of binding to apolypeptide selected from the group consisting of the amino acidsequences set forth by residues 37-47, 39-49, 41-49, 50-60, 58-68,80-90, 97-107, 50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137,55-102, 50-107, and 41-137 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM agonist is capable of binding to apolypeptide having at least about 80%, more preferably 85%, morepreferably 90%, more preferably 95% identity to the amino acid sequenceset forth by residues 31-41, 32-42, 35-43, 44-54, 52-62, 74-84, 88-98,44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 49-93, 44-98, 35-98,and 35-128 of the human BTLA isoform found at Genbank accession no.AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM agonist is capable of binding to apolypeptide selected from the group consisting of the amino acidsequences set forth by residues 31-41, 32-42, 35-43, 44-54, 52-62,74-84, 88-98, 44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 49-93,44-98, 35-98, and 35-128 of the human BTLA isoform found at Genbankaccession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM agonist is capable of competing with CMVUL144 for binding to BTLA.

In one embodiment, a BTLA-HVEM agonist is a BTLA antibody.

In one aspect, the invention provides BTLA-HVEM agonists that comprisean HVEM CRD1 domain or fragment thereof capable of binding to a BTLA Igdomain and stimulating BTLA activity. In another aspect, the inventionprovides BTLA-HVEM agonists that consist essentially of an HVEM CRD1domain or fragment thereof capable of binding to a BTLA Ig domain andstimulating BTLA activity.

Accordingly, in a preferred embodiment, the invention provides BTLA-HVEMagonists that are agonistic HVEM fusion proteins which are capable ofbinding to a BTLA Ig domain, reducing the binding of the BTLA Ig domainto an HVEM CRD1 domain, and stimulating BTLA activity. Such agonisticHVEM fusion proteins lack an HVEM CRD2 and/or CRD3 domain. Preferredagonistic HVEM fusion proteins can compete with a BTLA antibodydisclosed herein for binding to a BTLA Ig domain.

In another preferred embodiment, the invention provides BTLA-HVEMagonists that are agonistic HVEM protein fragments which are capable ofbinding to a BTLA Ig domain, reducing the binding of the BTLA Ig domainto an HVEM CRD1 domain, and stimulating BTLA activity. Such agonisticHVEM protein fragments lack an HVEM CRD2 and/or CRD3 domain. In apreferred embodiment, an agonistic HVEM protein fragment consistsessentially of an HVEM CRD1 domain of fragment thereof which is capableof binding to a BTLA Ig domain and stimulating BTLA activity. Preferredagonistic HVEM protein fragments can compete with a BTLA antibodydisclosed herein for binding to a BTLA Ig domain.

In one embodiment, a BTLA-HVEM agonist is capable of increasing tyrosinephosphorylation on the intracellular domain of BTLA protein in a cellhaving BTLA protein on its surface.

In one embodiment, a BTLA-HVEM agonist is capable of increasingassociation of BTLA protein with SHP-2, PI3K, or Grb2 in a cell havingBTLA protein on its surface.

In one embodiment, a BTLA-HVEM agonist is capable of decreasingproliferation of a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM agonist is capable of decreasing IL-2production by a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM agonist is capable of decreasing antibodyproduction by a cell having said BTLA protein on its surface.

In one embodiment, a BTLA-HVEM antagonist is capable of decreasing thecytotoxicity of a cell having said BTLA protein on its surface.

In one aspect, the invention provides methods for modulating BTLAactivity which involve the use of BTLA-HVEM agonists or BTLA-HVEMantagonists described herein.

In one aspect, the invention provides methods for decreasing BTLAactivity, comprising contacting BTLA or HVEM with a BTLA-HVEMantagonist.

In a preferred embodiment, the method comprises contacting a cell havingBTLA on its surface with a BTLA-HVEM antagonist, wherein the cell iscapable of contacting HVEM protein in the absence of the BTLA-HVEMantagonist. In one embodiment, the methods involve use of a BTLAantibody.

In a preferred embodiment, the cells having BTLA on their surface arelymphocytes, NK cells, or macrophages.

In another embodiment, the method comprises contacting HVEM protein witha BTLA-HVEM antagonist, wherein the HVEM protein is capable ofcontacting a cell having BTLA on its surface in the absence of theBTLA-HVEM antagonist. In one embodiment, the methods involve use of anHVEM antibody.

In a preferred embodiment, the HVEM protein is on the surface of adendritic cell or a lymphocyte.

In another embodiment, the invention provides methods for decreasingBTLA activation by a BTLA ligand that is capable of competing with HVEMfor binding to BTLA, which comprise the use of a BTLA-HVEM antagonist.In one embodiment, the BTLA ligand is CMV UL144.

In one aspect, the invention provides methods for increasing BTLAactivity comprising contacting a cell having BTLA on its surface with aBTLA-HVEM agonist. In one embodiment, the methods involve use of a BTLAantibody.

In a preferred embodiment, the cells having BTLA on their surface arelymphocytes, NK cells, or macrophages.

In one aspect, the invention provides methods for modulating lymphocyteactivation which involve the use of BTLA-HVEM agonists or BTLA-HVEMantagonists described herein.

In one aspect, the invention provides methods for increasing lymphocyteactivation. In one embodiment, the methods comprise contacting alymphocyte having BTLA on its surface with a BTLA-HVEM antagonist,wherein the lymphocyte is capable of contacting HVEM protein in theabsence of the BTLA-HVEM antagonist. In one embodiment, the methodsinvolve use of a BTLA antibody.

In one embodiment, the methods involve contacting HVEM protein with aBTLA-HVEM antagonist, wherein the HVEM protein is capable of contactinga lymphocyte having BTLA on its surface in the absence of the BTLA-HVEMantagonist. In one embodiment, the methods involve use of a HVEMantibody.

In a preferred embodiment, a lymphocyte in which activation is increasedis selected from the group consisting of naïve T cells, CD8⁺ T cells,CD4⁺ T cells, T_(H)1 cells, naive B cells, and plasma cells.

In another embodiment, the invention provides methods for decreasinglymphocyte activation, comprising contacting a lymphocyte having BTLA onits surface with a BTLA-HVEM agonist. In one embodiment, the methodsinvolve use of a BTLA antibody.

In a preferred embodiment, a lymphocyte in which activation is decreasedis selected from the group consisting of naïve T cells, CD8⁺ T cells,CD4⁺ T cells, T_(H)1 cells, naive B cells, and plasma cells.

In one aspect, the invention provides methods for modulating lymphocyteeffector activity.

In one aspect, the invention provides methods for decreasing lymphocyteeffector activity, comprising contacting a lymphocyte having BTLA on itssurface with a BTLA-HVEM agonist. In one embodiment, the methods involvethe use of a BTLA antibody. Decreasing lymphocyte effector activityincludes promoting the termination of effector activity, i.e.,shortening the duration of effector activity.

In one aspect, the invention provides methods for increasing and/orprolonging lymphocyte effector activity, comprising contacting alymphocyte having BTLA on its surface with a BTLA-HVEM antagonist. Inone embodiment, the methods involve the use of a BTLA antibody. Inanother embodiment, the methods involve contacting an HVEM protein witha BTLA-HVEM antagonist. Prolonging effector activity includes delayingthe termination of effector activity.

In another aspect, the invention provides methods for modulating animmune response to an antigen, which involve the use of BTLA-HVEMagonists or BTLA-HVEM antagonists described herein.

In one aspect, the invention provides methods for increasing an immuneresponse to an antigen, comprising contacting a lymphocyte having BTLAon its surface with a BTLA-HVEM antagonist, wherein the lymphocyte hasspecificity for the antigen, and wherein the lymphocyte is capable ofcontacting HVEM protein in the absence of the BTLA-HVEM antagonist. Inone embodiment, the methods involve use of a BTLA antibody.

In another embodiment, the methods comprise contacting HVEM protein witha BTLA-HVEM antagonist, wherein the HVEM protein is capable ofcontacting a lymphocyte having BTLA on its surface in the absence of theBTLA-HVEM antagonist, wherein the lymphocyte has specificity for theantigen. In one embodiment, the methods involve use of a HVEM antibody.

In a preferred embodiment, the antigen is a cancer cell antigen.

In another preferred embodiment, the antigen is a viral antigen.

In another preferred embodiment, the antigen is presented by a pathogen.

In another preferred embodiment, the antigen is provided by a vaccine.

In a preferred embodiment, the lymphocyte having BTLA on its surface andspecificity for the antigen is contacted with a BTLA-HVEM antagonist invivo.

In a preferred embodiment, the HVEM protein is contacted with aBTLA-HVEM antagonist in vivo.

In a preferred embodiment, the lymphocyte having specificity for theantigen is selected from the group consisting of naïve T cells, CD8⁺ Tcells, CD4⁺ T cells, T_(H)1 cells, naive B cells, and plasma cells.

In one embodiment, the methods further comprise administering antigen toa patient receiving the BTLA-HVEM antagonist.

In one embodiment, the methods further comprise administering abioactive agent that increases a positive costimulatory signal to apatient receiving the BTLA-HVEM antagonist.

In one embodiment, the methods further comprise administering abioactive agent that decreases a negative costimulatory signal to apatient receiving the BTLA-HVEM antagonist. For example, it iscontemplated that use of a BTLA-HVEM antagonist will be synergistic incombination with agents capable of providing CTLA-4 blockade asdescribed in U.S. Pat. Nos. 5,855,887; 5,811,097; and 6,051,227, andInternational Publication WO 00/32231, the disclosures of which areexpressly incorporated herein by reference.

In one embodiment, the invention provides methods for increasing animmune reaction against a tumor in a patient, comprising contacting alymphocyte having BTLA on its surface with a BTLA-HVEM antagonist,wherein the lymphocyte has specificity for a cancer cell antigenassociated with the tumor and is capable of contacting HVEM protein. Inone embodiment, the methods involve use of a BTLA antibody.

In another embodiment, the methods comprise contacting HVEM protein witha BTLA-HVEM antagonist, wherein the HVEM protein is capable ofcontacting a lymphocyte having BTLA on its surface, and wherein thelymphocyte has specificity for a cancer cell antigen associated with thetumor. In one embodiment, the methods involve use of a HVEM antibody.

In a preferred embodiment, the methods further comprise administering acancer cell antigen to the patient.

In a preferred embodiment, the methods further comprise administering abioactive agent that increases a positive costimulatory signal.

In a preferred embodiment, the methods further comprise administering abioactive agent that decreases a negative costimulatory signal to thecancer patient. For example, it is contemplated that use of a BTLA-HVEMantagonist will be synergistic in combination with agents capable ofproviding CTLA-4 blockade as described in U.S. Pat. Nos. 5,855,887;5,811,097; and 6,051,227, and International Publication WO 00/32231.

In a preferred embodiment, the lymphocyte having BTLA on its surface andspecificity for the cancer cell antigen is contacted with a BTLA-HVEMantagonist in vivo.

In a preferred embodiment, the HVEM protein is contacted with aBTLA-HVEM antagonist in vivo.

In a preferred embodiment, the lymphocyte having specificity for thecancer cell antigen is selected from the group consisting of naïve Tcells, CD8⁺ T cells, CD4⁺ T cells, T_(H)1 cells, naive B cells, andplasma cells.

In one aspect, the invention provides methods for inhibiting tumorgrowth, comprising administering to a patient a therapeuticallyeffective amount of a BTLA-HVEM antagonist.

In a preferred embodiment, the methods further comprise administering acancer cell antigen to the patient.

In a preferred embodiment, the methods further comprise administering abioactive agent that increases a positive costimulatory signal.

In a preferred embodiment, the methods further comprise administering abioactive agent that decreases a negative costimulatory signal to thecancer patient. For example, it is contemplated that use of a BTLA-HVEMantagonist will be synergistic in combination with agents capable ofproviding CTLA-4 blockade as described in U.S. Pat. Nos. 5,855,887;5,811,097; and 6,051,227, and International Publication WO 00/32231.

In one aspect, the invention provides methods for treating cancer,comprising administering to a patient a therapeutically effective amountof a BTLA-HVEM antagonist.

In a preferred embodiment, the methods further comprise administering acancer cell antigen to the patient.

In a preferred embodiment, the methods further comprise administering abioactive agent that increases a positive costimulatory signal.

In a preferred embodiment, the methods further comprise administering abioactive agent that decreases a negative costimulatory signal to thecancer patient. For example, it is contemplated that use of a BTLA-HVEMantagonist will be synergistic in combination with agents capable ofproviding CTLA-4 blockade as described in U.S. Pat. Nos. 5,855,887;5,811,097; and 6,051,227, and International Publication WO 00/32231.

In one aspect, the invention provides methods for reducing an immuneresponse to an antigen, comprising contacting a lymphocyte having BTLAon its surface with a BTLA-HVEM agonist, wherein the lymphocyte hasspecificity for the antigen. In one embodiment, the methods involve useof a BTLA antibody.

In a preferred embodiment, the antigen is a graft cell antigen.

In another preferred embodiment, the antigen is a self antigen.

In another preferred embodiment, the lymphocyte having specificity forthe antigen is selected from the group consisting of naïve T cells, CD8⁺T cells, CD4⁺ T cells, T_(H)1 cells, naive B cells, and plasma cells.

In one embodiment, the methods further comprise administering abioactive agent that decreases a positive costimulatory signal to thepatient.

In one embodiment, the methods further comprise administering animmunosuppressant to the patient.

In one embodiment, the methods further comprise administering abioactive agent that increases a negative costimulatory signal to thepatient.

In one embodiment, the invention provides methods for reducing an immunereaction against a graft in a patient, comprising contacting alymphocyte having BTLA on its surface with a BTLA-HVEM agonist, whereinthe lymphocyte has specificity for a graft cell antigen. In oneembodiment, the BTLA-HVEM agonist is a BTLA antibody.

In another preferred embodiment, the lymphocyte having specificity forthe antigen is selected from the group consisting of naïve T cells, CD8⁺T cells, CD4⁺ T cells, T_(H)1 cells, naive B cells, and plasma cells.

In one embodiment, the methods further comprise administering abioactive agent that decreases a positive costimulatory signal to thepatient.

In one embodiment, the methods further comprise administering animmunosuppressant to the patient.

In one embodiment, the methods further comprise administering abioactive agent that increases a negative costimulatory signal to thepatient.

In one aspect, the invention provides methods for reducing rejection ofa graft by a patient, comprising administering to the patient atherapeutically effective amount of a BTLA-HVEM agonist.

In one embodiment, the methods further comprise administering abioactive agent that decreases a positive costimulatory signal to thepatient.

In one embodiment, the methods further comprise administering animmunosuppressant to the patient.

In one embodiment, the methods further comprise administering abioactive agent that increases a negative costimulatory signal to thepatient.

In one aspect, the invention provides methods for prolonging thesurvival of a graft in a patient, comprising administering to thepatient a therapeutically effective amount of a BTLA-HVEM agonist.

In one embodiment, the methods further comprise administering abioactive agent that decreases a positive costimulatory signal to thepatient.

In one embodiment, the methods further comprise administering animmunosuppressant to the patient.

In one embodiment, the methods further comprise administering abioactive agent that increases a negative costimulatory signal to thepatient.

In one aspect, the invention provides methods for reducing a graftversus host response in a patient, comprising administering to thepatient a therapeutically effective amount of a BTLA-HVEM antagonist.

In one embodiment, the methods further comprise administering abioactive agent that increases a positive costimulatory signal to thepatient.

In one embodiment, the methods further comprise administering abioactive agent that decreases a negative costimulatory signal to thepatient. For example, it is contemplated that use of a BTLA-HVEMantagonist will be synergistic in combination with agents capable ofactivating CTLA-4 as described in U.S. Pat. Nos. 5,855,887; 5,811,097;and 6,051,227, and International Publication WO 00/32231.

In one aspect, the invention provides methods for treating a patienthaving an autoimmune disease, comprising administering to the patient atherapeutically effective amount of a BTLA-HVEM agonist.

In one embodiment, the autoimmune disease is selected from the groupconsisting of Rheumatoid arthritis, type 1 diabetes, autoimmunethyroiditis, and Lupus.

In one embodiment, the methods further comprise administering abioactive agent that decreases a positive costimulatory signal to thepatient.

In one embodiment, the methods further comprise administering animmunosuppressant to the patient.

In one embodiment, the methods further comprise administering abioactive agent that increases a negative costimulatory signal to thepatient.

In one aspect, the invention provides methods for treating a patienthaving an allergic reaction, comprising administering to the patient atherapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for preventing a patientfrom having an allergic reaction, comprising administering to thepatient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for reducing an allergicreaction in a patient, comprising administering to the patient atherapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for reducing an asthmaticresponse in a patient, comprising administering to the patient atherapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for enhancing recoveryfrom an asthmatic response in a patient, comprising administering to thepatient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for treating asthma,comprising administering to an asthma patient a therapeuticallyeffective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for reducing aninflammatory reaction in a patient, comprising administering to thepatient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for reducing theinteraction of cell having BTLA on its surface and a second cell havingHVEM on its surface. The methods involve the use of a BTLA-HVEMantagonist or a BTLA-HVEM agonist. In a preferred embodiment, themethods involve use of a BTLA antibody or a HVEM antibody. In apreferred embodiment, the cell having BTLA on its surface is selectedfrom the group consisting of naïve T cells, CD8⁺ T cells, CD4⁺ T cells,T_(H)1 cells, naive B cells, and plasma cells.

In one aspect, the invention provides methods for modulating memory cellformation, comprising contacting a lymphocyte exposed to antigen with aBTLA-HVEM agonist or antagonist. In a preferred embodiment, the methodsinvolve the use of a BTLA antibody.

In one aspect, the invention provides methods for modulating toleranceof self antigen, comprising contacting a lymphocyte exposed to selfantigen with a BTLA-HVEM agonist or antagonist. In a preferredembodiment, the methods involve the use of a BTLA antibody.

Also provided are adjuvant compositions comprising at least one of theBTLA-HVEM antagonists described herein.

Also provided are immunosuppressant compositions comprising at least oneof the BTLA-HVEM agonists described herein.

In another aspect, the present invention provides methods of screeningfor BTLA-HVEM agonists and BTLA-HVEM antagonists, which agonists andantagonists find therapeutic uses for the modulation of immunereactions.

The invention further contemplates the use of the aforementionedpolypeptides in immunoassays.

The invention further contemplates the use of the aforementionedpolypeptides as immunogens for the production of antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 BTLA recognizes a ligand on naive T cells. Splenocytes fromBALB/c and C57BL/6 mice were collected and either were directly stained(None) or were activated with plate-bound 500A2 (Anti-CD3; 1:200dilution of ascites fluid) or soluble anti-IgM (10 μg/ml) for 48 h, andthen were stained with BTLA-Fc or PD-L1-Fc fusion protein (shadedhistograms) followed by anti-human IgG-phycoerythrin (Anti-humanIgG-PE), anti-CD4-tricolor and anti-B220-FITC. Open histograms, stainingwith human IgG1 isotype control in place of Fc fusion protein.

FIG. 2 BTLA tetramer staining identifies a ligand on CD4+ and CD8+cells. (a) Splenocytes and lymph node cells from pooled C57BU6 andBALB/c mice were stained with anti-CD8-FITC, anti-CD4-CyChrome, andeither streptavidin-phycoerythrin (open histograms) or BTLAtetramer-phycoerythrin (shaded histograms). Dot plots (left) show theCD4-CD8 gates used for single-color histograms of BTLAtetramer-phycoerythrin staining (right). (b) Splenocytes from pooledC57BU6 and BALB/c mice were left untreated or were activated 48 h withanti-CD3 or anti-IgM as described in FIG. 1 or with lipopolysaccharide(1 μg/ml) for 24 h and were stained with anti-B220-FITC,anti-CD4-CyChrome, and either streptavidin-phycoerythrin (openhistograms) or BTLA tetramer-phycoerythrin (shaded histograms). (c)Thymocytes from pooled C57BU6 and BALB/c mice were stained withanti-CD8-FITC, anti-CD4-CyChrome, and either streptavidin-phycoerythrin(open histograms) or BTLA tetramer-phycoerythrin (shaded histograms).The dot plot (left) shows the CD4/CD8 gates used for the single-colorhistograms of BTLA-tetramer staining.

FIG. 3 BTLA ligand expression is modulated during T cell activation.DO11.10 splenocytes were stimulated with 0.3 μM OVA peptide in T helpertype 1 conditions (T_(H)1; 10 U/ml of IL-12 and 10 μg/ml of anti-IL-4)or T helper type 2 conditions (T_(H)2; 100 U/ml of IL-4 and 3 μg/ml ofanti-IL-12). Cultures were collected after activation (time, horizontalaxis) and were stained with anti-CD4-FITC and BTLAtetramer-phycoerythrin. Filled circles, streptavidin-phycoerythrin(SA-PE) staining of T helper type 1 cultures without BTLA tetramer. MFI,mean fluorescence intensity.

FIG. 4 HVEM is a ligand for BTLA. (a) NIH 3T3 cells and BJAB cells weretransduced with splenocyte cDNA libraries and were directly stained withanti-Thy1.1-FITC and the C57BL/6 BTLA tetramer-phycoerythrin (Beforesorting). These cells were sorted for the highest 0.5% population ofBTLA tetramer staining with BTLA-phycoerythrin tetramer and Thy1.1-FITCand were subjected to an additional three rounds of similar sequentialpurification. After the fourth round of sorting, cell populations wereexpanded and cells were stained (After sorting). Numbers in eachquadrant indicate the percentage of live cells in the indicated gate.(b) BJAB cells were transduced with the retroviruses mHVEM-IRES-GFP(mHVEM; mouse), hHVEM-IRES-GFP (hHVEM; human), 4-1 BB-IRES-GFP (4-1BB;mouse) and LTβR-IRES-GFP (LTβR; mouse) and were stained with C57BL/6BTLA tetramer-phycoerythrin or BALB/c BTLA tetramer-phycoerythrin.Numbers in dot plots indicate the percentage of BTLA tetramer stainingin the GFP-positive population. (c,d) HVEM activates BTLAphosphorylation and SHP-2 association. EL-4 cells (EL4), BJAB cellsexpressing GFP (BJAB-GFP) or BJAB cells expressing mouse HVEM(BJAB-mHVEM) were added (+) or not added (−) for 4 min at 37° C. at adensity of 25×10⁶ cells/ml. Cells were left untreated (−) or weretreated (+) with pervanadate (VO₄) for 4 min. Total cell lysates wereprepared and were immunoprecipitated with 6A6 (anti-mouse BTLA), andimmunoblots were probed for SHP-2 (c) or for phosphotyrosine (d) inimmunoprecipitates (IP) or in lysates without immunoprecipitation.Immunoblots using the isotype control for immunoprecipitation werenegative for SHP-2 association (data not shown). Data in c and d arerepresentative of four independent experiments. (e) BJAB cells weretransduced with retrovirus mHVEM-ires-GFP or hHVEM-ires-GFP and werestained with human IgG1 isotype control (hIgG1), mB7x-Fc, mBTLA-Fc orhBTLA-Fc followed by anti-human IgG-phycoerythrin. Numbers in dot plotsshow the percentage of fusion protein staining in the GFP-positivepopulation.

FIG. 5 HVEM is the unique ligand for BTLA and interacts through CRD1.(a) Splenocytes from wild-type (Tnfrsf14+/+) or HVEM-deficient(Tnfrsf14−/−) mice were stained with anti-CD4-FITC (CD4+), anti-CD8-FITC(CD8+) or anti-B220-FITC (B220+) and either C57BL/6 BTLAtetramer-phycoerythrin (shaded histograms) or streptavidin-phycoerythrinalone (open histograms). (b) Splenocytes from wild-type (Btla+/+) andBtla−/− mice were stained with anti-B220-FITC (top) or anti-CD11c-FITC(bottom) and with either mHVEM-Fc (shaded histograms) or isotype controlhuman IgG1 (open histograms) followed by anti-human IgG-phycoerythrin.(c) Splenocytes from wild-type (Tnfsf14+/+) and Tnfsf14−/− mice werestained with B220-FITC (top) or CD11c-FITC (bottom) and mHVEM-Fc orisotype control human IgG1 (open histograms), followed by anti-humanIgG-phycoerythrin. (d) BJAB cells were left uninfected or weretransduced with retroviruses expressing mouse HVEM-GFP fusion protein(mHVEM-GFP), the HVEM deletion mutant lacking N-terminal CRD1 as a GFPfusion protein (m/hVEMCRD1-GFP), intact human HVEM (hHVEM-IRES-GFP) orchimeric HVEM containing mouse CRD1 linked to human CRD2(m/hHVEM-IRES-GFP). Left, cells stained with BTLA tetramer-phycoerythrin(shaded histograms) or streptavidin-phycoerythrin alone (openhistograms); right, cells stained with either anti-hHVEM (shadedhistograms) or a mouse IgG1 isotype control (9E10) followed by goatanti-mouse IgG1-phycoerythrin. Single-color histograms were gated onGFP-positive live cells. Right margin, composition of the HVEMconstructs, with mouse CRDs (open ovals) and human CRDs (shaded ovals).

FIG. 6 HVEM expression on APCs inhibits T cell proliferation. (a) CD4+cells were purified from BALB/c mice by magnetic separation and werestimulated (1×10⁶ cells/ml) with plate-bound anti-CD3 (2C11; dose,horizontal axis) and increasing concentrations (wedges; 0, 0.3, 1.0, 3.0and 10.0 μg/ml) of plate-bound LIGHT. Cultures were pulsed with[³H]thymidine at 48 h and were collected at 60 h. Data represent c.p.m.s.d. from one of three similar experiments. (b) CD4+ T cells fromDO11.10 mice were purified by magnetic separation, followed by cellsorting for CD4+ B220-CD11c-cells to more than 98% purity, and wereadded to cultures alone (T alone) or with (T+) CHO cells expressingI-Ad, I-Ad and B7.1, or I-Ad and BTLA, plus various concentration of OVApeptide (horizontal axis), and proliferation was measured as describedin a. (c) T cells prepared as described in b were cultured alone or withCHO cells expressing I-Ad, or I-Ad and HVEM, plus various concentrationsof OVA peptide, and proliferation was measured as described in a. (d) Tcells prepared as described in b were cultured alone or with CHO cellsexpressing I-Ad, or I-Ad and B7.1, or I-Ad, B7.1 and HVEM, and wereactivated with various concentration of OVA peptide. Proliferation wasmeasured as described in a.

FIG. 7 HVEM inhibits T cell proliferation in a BTLA-dependent way. (a)Highly purified DO11.10 CD4+ T cells from wild-type (Btla+/+) or Btla−/−mice were prepared as described in FIG. 6, were labeled with CFSE andwere cultured for 3 or 4 d with CHO cells expressing I-Ad, or I-Ad andBTLA, or I-Ad and HVEM, plus 0.03 or 0.3 μM OVA peptide. Cells wereanalyzed by flow cytometry. Data are single-color histograms of CFSEgated on CD4+ T cells. Numbers indicate percentage of live cells thathave divided at least once, as indicated by the gate drawn. (b) T cellsprepared as described in a were cultured for 3 or 4 d with CHO cellsexpressing I-Ad and B7.1, I-Ad, B7.1 and BTLA, or I-Ad, B7.1 and HVEM,plus 0.03 or 0.3 μM OVA peptide, and were analyzed as described in a.Numbers indicate percentage of live cells that have divided at leastonce.

FIG. 8. Polymorphisms in the BTLA Ig domain. A, Exon 2 of BTLA,comprising the Ig domain, was amplified by PCR from genomic DNA of theindicated mouse strains and sequenced. The amino acid alignment of theIg domains of BALB/c (SEQ ID NO:3), MLR/lpr (SEQ ID NO:4), and C57BL/6(SEQ ID NO:5) BTLA is shown, starting with the aspartic acid (D) residuethat corresponds to residue 37 of the entire BTLA protein (15). The lastline of the alignment shows a consensus sequence (bottom), withdifferences between BALB/c and MLR/lpr (#) and differences betweenBALB/c and C57BL/6 (*) shown. B, Strains sharing identical alleles ofBTLA are grouped together under the index headings of BALB/c, MLR/lpr,and C57BL/6.

FIG. 9. Production of mAbs to allelic variants of murine BTLA. A and B,BJAB cells were stably transfected with retroviral constructs expressingthe extracellular/transmembrane domains of BTLA from C57BL/6 (BJAB.B6BTLA-GFP, solid histogram) or BALB/c (BJAB.BALB/c BTLA-GFP, dottedhistogram) as GFP fusion proteins. Cells were stained with the indicatedpurified mAbs or postimmune serum (hamster anti-BTLA (A) serum, mouseanti-BTLA (B) serum). Secondary staining was with either anti-hamsterIgG (A) or anti-mouse Ig (B). Histograms shown are gated on GFP+ BJAB.B6 BTLA-GFP or BJAB.BALB/c BTLA-GFP cells stained separately. Shadedhistogram for the hamster and mouse immune serum are controls usingnormal hamster serum or normal mouse serum to stain a mixture of BJAB.B6 BTLA-GFP and BJAB.BALB/c BTLA-GFP cells. Shaded histogram for mAbstaining shows the isotype control of either hamster IgG (A) or murineIgG1 (B) staining a mixture of cells. C, Splenocytes from C57BL/6 orBALB/c wild-type mice (solid histogram) or BTLA−/− mice (dottedhistogram) were stained with 6A6 (left) or 6F7 (right). BTLA−/− stainingwas equivalent to that of the isotype control (shaded histogram). D,Lysates from 25×106 cells BJAB. B6 BTLA-GFP or BJAB.BALB/c BTLA-GFPcells were immunoprecipitated (IP) with 10 μg of the indicated Ab andWestern blots probed (Blot) with either 6F7, or with anti-GFP Ab, asindicated. As controls, cell lysates were immunoprecipitated with mouseor hamster IgG as indicated (lanes 7-10). E, EL4 cells were incubated inthe absence (−) or presence (+) of pervanadate for 4 min at 37° C., andlysed in 1% Triton X-100 lysis buffer, immunoprecipitated (IP) with 6A6or isotype control Ab (PIP anti-GST) and Western blots probed (Blot)with anti-SHP-2 as described.

FIG. 10. Mapping epitopes recognized by BTLA Abs using Yeast Display. Apanel of yeast cells expressing the indicated BTLA Ig domain Aga2 fusionproteins was analyzed for Ab staining. As a positive control, expressionof the fusion protein was confirmed first for each line using stainingwith anti-HA Ab specific for the HA-tag incorporated into the BTLA-Aga2fusion protein, and was positive for each line tested (data not shown).Yeast cells were stained with the anti-BTLA Ab indicated on top of eachcolumn. The amino acid substitutions (and corresponding nucleotidesubstitutions) in each yeast line are indicated on the left.Single-color histograms are marked (*) to indicate mutations that arenot recognized by the corresponding Ab.

FIG. 11. BTLA shows broad and allelic-specific expression on lymphoidcell populations. A, Four-color FACS analysis was conducted onsplenocytes from C57BL/6 (solid histogram) or BALB/c (dotted histogram).Two-color histograms (upper row) of the indicated markers used to gatecells for single-color histograms of 6A6 (middle row) or 6F7 (lower row)staining are shown. In the columns one, two, and three, cells werestained with anti-B220 allophycocyanin, anti-CD4 CyChrome, anti-CD8FITC, and either biotinylated b-6A6 or b-6F7 followed by SA-PEsecondary. In columns four, five, and six, cells were stained withanti-I-Ad PE (BALB/c cells) or anti-I-Ab PE (C57BU6 cells), andanti-CD11b FITC (fourth column), CD11c-FITC (fifth column), or anti-DX-5FITC (sixth column), and b-6A6 or b-6F7 followed by SA-CyChromesecondary. Shaded histograms are staining of a mixture of C57BL/6 andBALB/c splenocytes using isotype controls of biotinylated hamster IgG(middle row) and mouse IgG1 (lower row). The numbers shown in top panelsare the percentage of live cells within the indicated gate. The identityof the gated population is indicated in the panel. B, C57BL/6 and BALB/csplenocytes were stained with Abs to identify the following B cellpopulations: follicular B cells (FO), IgMlowCD21/CD35int; marginal zone(MZ), IgMhighCD21/CD35high; transitional (TR), IgMlowCD21/CD35low.Staining with the pan-BTLA-specific Ab 6F7 revealed equivalent BTLAlevels between strains for all subsets.

FIG. 12. BTLA is expressed during late stages of B and T lymphocytedevelopment. A, Thymocytes from C57BL/6 (solid histogram) or BALB/c(dotted histogram) mice were stained with a combination of markers,anti-B220 FITC, anti-CD11c FITC, anti-CD11b FITC, anti-GR-1 FITC,anti-DX-5 FITC, CD4-CyChrome, CD8-PE, and either biotinylated (b)-6A7 orb-mouse IgG1, and SA-allophycocyanin. The two-color histogram (firstpanel) is gated on marker (FITC)-negative live cells, and the numbersindicate the percentage of cells in the indicated gates. Single-colorhistograms for each gate are shown for b-6F7/SA-allophycocyanin stainingfor CD4−CD8− double negative (DN), CD4+CD8+ double positive (DP), CD4+single positive (CD4 SP), or CD8+ single positive (CD8 SP) populations.Shaded histograms are staining the b-mouse IgG1 isotype control. B, Bonemarrow cells were stained with anti-B220 allophycocyanin, anti-IgM PerCpCy5.5, either b-6F7 or murine IgG1-biotin, and SA-PE. The numbers arethe percentage of live gated cells within the three numbered gates. BTLAexpression is shown in the single-color histograms for each gate; gate1, Pre-B cells and Pro-B cells (IgM-B220low); gate 2, Immature B cells(IgM+B220low); gate 3, Mature B cells (IgM+B220high). Shaded regions aremouse IgG1 isotype control staining.

FIG. 13. BTLA expression during CD4+ T cell activation and Th1polarization. A, DO11.10 transgenic T cells were purified by cellsorting and activated with 0.3 μM OVA peptide 324-336 under Th1 or Th2conditions (see Examples). Cells were harvested either before activation(Day 0) or on the indicated day following primary activation, andstained with KJ1-26 Tricolor, b-6F7, and SA-PE. T cells wererestimulated with OVA peptide on day 7 and day 14. B, BALB/c splenocyteswere stimulated with 10 μg/ml anti-1 μM and 5 μg/ml anti-CD40 (left) or1 μg/ml LPS (right). Single-color histograms of B220+ cells(anti-B220-FITC) are shown for b-6F7/SA-PE staining on day 0 (dottedhistogram) and day 2 (solid histogram) after activation. Shadedhistograms are the biotinylated mouse IgG1 isotype control.

FIG. 14. BTLA is induced on anergic CD4+ T cells, but not CD4+ CD25+regulatory T cells. A, HA-TCR T cells were transferred into andsubsequently harvested from B10.D2 mice (naive), C3-HAhigh mice(anergized) or B10.D2 mice infected with vaccinia-HA (activated) on days2, 3, 4, or 7 after transfer as indicated. After harvest, T cells wereisolated using combined magnetic bead and fluorescence sorting, and cDNAprobe prepared and hybridized to Affymetrix microarrays M174A, M174B,and M174C. Relative BTLA expression intensity was determined using alatin-squares approach in Affymetrix Microarray Suite, version 5.1.software. Expression of myosin Vila gene is shown as a control. B,CFSE-labeled HA-TCR T cells were adoptively transferred into B10.D2 mice(naive), C3-HAhigh mice (anergized), or B10.D2 mice immunized withvaccinia-HA (activated), and harvested on day 6 as in A. Cells werestained with anti-CD4 allophycocyanin, anti-Thy1.1 PerCP, and eitherb-6F7 or murine IgG1-biotin, and SA-PE. BTLA expression is shown assingle-color histogram for CFSE+(naive) or CFSE-(activated andanergized) for CD4+ Thy1.1+ donor cells. C, Splenocytes harvested fromrecipients as in A were restimulated with HA peptide and proliferationmeasured on day 2. D, Splenocytes and lymph node cells from BALB/c micewere enriched for CD25-negative and CD25-positive populations usinganti-CD25-PE and magnetic beads as described in Materials and Methods,and stained with anti-CD4-Cy-chrome, and biotin-conjugated 6F7, orbiotin-IgG1, followed by SA-allophycocyanin. Two-color dot plots areshown for CD25 and CD4 (left panels), or single-color histograms gatedon CD4+ cells for 6F7 (middle panels) or anti-PD-1 (right panels) forthe CD25− (top row) and CD25+ (bottom row) fractions. For BTLA staining,histograms are shown for both the freshly isolated cells (thinhistogram) and 36 h anti-CD3-activated cells (thick histogram). Shadedhistograms are the staining of the mouse IgG1 istoype control. E, Cellsisolated in D were stimulated with the indicated amount of anti-CD3 andproliferation measured after 2 days.

FIG. 15. BTLA−/− mice have modestly augmented IgG3 responses toT-independent Ag. 129SvEv wild-type mice or BTLA−/− mice (n=5) wereimmunized with 50 μg NP-Ficoll in alum by i.p. injection. At day 14,relative isotype-specific anti-NP Ab titer in serum was determined byELISA. Data are shown as the percentage of the Ab titer produced inserum of naive BTLA+/+ or BTLA−/− mice. Mean±SD is shown.

FIG. 16 BTLA−/− parental cells engraft and initially expand

FIG. 17 BTLA−/− parental cells fail to survive following transfer

FIG. 18 BTLA−/− cells do not persist as GHVD progresses Until about day9, the expansion of WT and BTLA KO donor T cells is similar; At latertimes, BTLA−/− show rapid decrease is the number of remaining donorcells.

FIG. 19 HVEM induces BTLA-phosphorylation and SHP-2 recruitment intrans.

FIG. 20 HVEM on APCs inhibits T cell proliferation through BTLA. HVEM onAPCs inhibits T cell proliferation. HVEM does not inhibit BTLA−/− Tcells.

FIG. 21 HVEM on APCs inhibits T cell proliferation through BTLA.

FIG. 22 HVEM inhibition is overcome by strong costimulation. HVEMinhibition of T cells is less with stronger co-stimulation. HVEMinhibition of T cells is less at highest antigen doses.

FIG. 23 6A6 binds to amino acid residues E34 and R73 of BTLA. Antibodyinteractions are most affected by E34Q and R73Q mutations, and slightlyaffected by H23Q and W56C mutations. E 34 and R73 are E63 and R102 infull length protein.

FIG. 24 shows the amino acid sequence of human BTLA, also found atGenbank Accession No. AAP44003.1 (SEQ ID NO:2).

FIG. 25 PD-1 and BTLA are expressed on BAL CD4 T cells: C57BU6 mice weresensitized and challenged with Ovalbumin. On days 1, 3, 4, and 7following challenge, groups of mice were euthanized and the cellsrecovered in the BAL analyzed for expression of CD4 and PD-1 or BTLA by2-color flow cytometry. The percentage of cells positive for CD4 as afraction of either the total sample or of the lymphocyte gate as well asthe total number of CD4+ cells recovered is indicated in each box.Histograms of PD-1 or BTLA expression on the CD4+ cells are shown fordays 3, 4 and 7. Representative data of 3 independent experiments ispresented.

FIG. 26 PD-1 and BTLA have a minor effect on acute allergic airwayinflammation: C57BU6, PD-1−/− and BTLA−/− mice (n=5 per group) weresensitized ad challenged with OVA. 3 days following challenge, the micewere euthanized and samples collected for analysis. A) Total cell countsin the BAL fluid. B) Differential analysis of the cell types present inthe BAL. C) Representative fields of H and E stained sections (40×magnification). *=P<0.05**=P<0.005 compared to C57BU6 by 2 tailed Ttest. Representative data from 5 independent experiments is shown.

FIG. 27 Expression of the ligands for PD-1 and BTLA during allergicairway inflammation: Total RNA was isolated from whole lungs of allergenchallenged mice on the indicated days post-challenge or from primarycultured murine tracheal epithelial cells (mTEC). RT-PCR was performedusing specific primers that spanned intronic sequences of each gene.Shown is representative data from 2 independent experiments.

FIG. 28 shows that PD-1 and BTLA-deficient mice have a prolongedduration of airway inflammation: C57BL/6, PD-1−/− and BTLA−/− mice weresensitized and challenged with OVA. On days 10 and 15 cohorts of mice(n=5/group) were euthanized and samples collected for A) analysis of theBAL and B) histology. *=p<0.05 compared to C57BL/6 using a 2 tailedT-test.

FIG. 29 shows graphs and micrographs illustrating that BTLA and HVEM,but not PD-1, regulate the survival of partially MHC-mismatched cardiacallografts. a, The lack of BTLA or HVEM, or administration of aneutralizing anti-BTLA mAb, led to rejection of all MHC classII-mismatched cardiac allografts within 3-4 wk of transplantation,whereas wild-type (WT) recipients accepted Bm12 allografts indefinitely.Data were generated from six to 12 allografts/group; p<0.001 forBTLA−/−, HVEM−/−, or anti-BTLA mAb-treated group vs respective WTcontrols. Panels at the right show acute cellular rejection of Bm12allografts harvested 2 wk after transplant from BTLA−/−, but not WT,recipients (H&E-stained paraffin sections; original magnifications,×300). b, In contrast to BTLA and HVEM, a lack of PD-1 stillallowed >80% long-term survival of MHC class II-mismatched cardiacallografts (p<0.05 compared with isotype-treated WT control), and anabsence of both PD-1 and BTLA (DKO) led to only a minor acceleration ofallograft rejection compared with lack of BTLA alone (p<0.05 vs BTLA−/−alone) in B6 recipients of Bm12 cardiac allografts. Data were generatedfrom four to eight allografts per group. c, Lack of BTLA led torejection of all MHC class I-mismatched cardiac allografts, whereas WTrecipients accepted Bm1 allografts indefinitely. Data were generatedfrom six to 12 allografts/group (p<0.001). Panels at the right showhistologic evidence of developing cellular rejection of Bm1 allograftsharvested 4 wk after transplant from BTLA−/−, but not WT, recipients(H&E-stained paraffin sections; original magnifications, ×300).

FIG. 30 shows graphs illustrating that BTLA suppresses T cell responsesto MHC class II alloantigens. a, Intragraft mRNA expression of BTLA,PD-1, and ligands was determined by qPCR; data are expressed as the foldincrease compared with naive heart and are representative of threeseparate experiments (Bm123B6 cardiac allografts). b, Compared withwild-type (WT) CD4⁺ T cells, CD4⁺ T cells from BTLA−/− mice had markedlyenhanced proliferative responses to Bm12 APC. Data at 72 h are expressedas a percentage of live BrdU⁺ CD4 cells at each stimulator (S) toresponder (R) ratio (pooled triplicate wells). c, Assessment ofalloactivation-induced CD4⁺ T cell proliferation at 72 h induced byirradiated Bm12 APC; the percentage of dividing CD4⁺ T cells wasdetermined by CFSE dilution. d, Markedly increased proliferation ofCFSE-labeled BTLA−/− CD4⁺ T cells 72 h after transfer into irradiatedBm12 hosts. Data are representative of two experiments with similarresults. e, Marginally increased proliferation of CFSE-labeled BTLA−/−CD8⁺ T cells 72 h after transfer into irradiated Bm12 hosts. Data arerepresentative of two experiments with similar results. f, Significantlyincreased responder frequency in BTLA−/− recipients of classII-mismatched cardiac allografts, as shown by harvesting of recipientspleens 10 days after transplant and stimulation of recipientsplenocytes in vitro with irradiated Bm12 (p<0.001 at all ratios) or B6DC (syngeneic control) for 24 h. Donor-specific responder frequency wasexpressed as the number of IFN-γ spot-forming cells (SFC) per 1×10⁶splenocytes, and data (mean±SD) are representative of two experiments.

FIG. 31 shows graphs and photographic images demonstrating that BTLAtargeting prolongs survival of fully MHC-mismatched cardiac allografts.Targeting of BTLA significantly prolonged BALB/c cardiac allograft infully allogeneic B6 recipients, as shown using BTLA−/− recipients (a)and anti-BTLA mAb in wild-type (WT) mice (b). c, In addition, asubtherapeutic course of rapamycin (RPM; 10 μg/kg/day, i.p., for 14days) significantly prolonged cardiac allograft survival compared witheither identically treated WT mice or BTLA−/− controls. Allograftsurvival data in a-c were obtained from six to eight transplants pergroup. d, BALB/c hearts transplanted to WT or BTLA−/− B6 mice wereharvested 7 days after transplant for qPCR. Data from three allograftsper group are expressed as the fold increase compared with native heart.e, Western blots of CXCR3 and IP-10 proteins, using extracts of threeallografts per group. The effects of targeting BTLA, alone or incombination with low dose RPM, on allogeneic T cell proliferation andcytokine production were determined by adoptive transfer of CFSE-labeledsplenocytes from WT or BTLA−/− mice to B6D2F1 hosts, and recipientspleens were harvested at 72 h. The responses of donor T cells wereidentified by gating on Kd−Dd− cells. Data are shown as an overlay ofCFSE histograms (f) and analysis of intracellular cytokine production(g). The figure in each box is the percentage of the indicatedpopulation, and data are representative of two experiments with similarresults.

FIG. 32 shows graphs illustrating the dominant role of PD-1 inregulating the survival of fully MHC-mismatched cardiac allografts. a,Dual PD-1/BTLA−/− (DKO) recipients rejected fully MHC-disparateallografts at the same speed as wildtype (WT) recipients. b,Neutralization of PD-1 in BTLA−/− recipients reversed the prolongationof survival seen in BTLA−/− mice (p<0.001). c, The dominant role of PD-1was also seen by the quick rejection of allografts in DKO mice, despitetherapy with rapamycin (RPM; 10 μg/kg/day, i.p., for 14 days), in markedcontrast to the prolonged survival in BTLA−/− recipients treated withthe same dose of RPM (p<0.001). d, The key contribution of PD-1, but notBTLA, in promoting the survival of fully MHC-mismatched cardiacallografts in RPM-treated recipients was confirmed by the rapid onset ofacute rejection in BTLA−/− recipients treated with anti-PD-1 mAb(p<0.001).

FIG. 33 shows plots that demonstrate increased PD-1 expression andfunction by alloreactive T cells of BTLA−/− recipients of fullyMHC-mismatched cardiac allografts. a, Intragraft mRNA expression ofBTLA, PD-1, and ligands was determined by qPCR. Data are expressed asthe fold increase compared with naïve heart and are representative ofthree separate experiments (BALB/c3B6 cardiac allografts). b, PD-1expression by alloreactive T cells determined by adoptive transfer ofCFSE-labeled wild-type (WT) or BTLA−/− splenocytes to irradiated Bm12 orB6D2F1 hosts, with or without added rapamycin (RPM; 0.01 mg/kg, i.p.,for 3 days). Figures indicate the percentages of PD-1_ cells in thedivided and undivided donor T cell populations. c, Increasedproliferation of CFSE-labeled T cells from DKO mice or PD-1−/− mice vsWT or BTLA−/− controls after adoptive transfer to F1 hosts, with orwithout RPM therapy. Analysis of corresponding intracellular cytokineproduction by the groups shown in c was undertaken, alone (d) or inconjunction with RPM therapy (e). Cells were stained with Kd−PE and CD4−or CD8−PerCP, and IL-2 or IFN-γ APCs and donor cells were identified asthe Kd−Dd− population; the percentage of each indicated population isshown.

FIG. 34 shows graphs illustrating that as the strength of T cellsignaling increases, PD-1 induction predominates over that of BTLA.Increasing T cell activation by mature fully allogeneic BALB/c bonemarrow-derived DC leads to increasing expression of PD-1, rather thanBTLA, by C57BL/6 CD4 and CD8 T cells, as shown by flow cytometricanalysis of cells cultured at varying stimulator (S) to responder (R)ratios for 72 h. Data are representative of three such experiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the term “HVEM CRD1 domain” refers to the CRD1 domain ofan HVEM protein. An HVEM CRD1 domain binds to a BTLA Ig domain, and canbe specifically bound by a preferred HVEM antibody disclosed herein. TheHVEM CRD1 domain does not include the CRD2 or CRD3 domains of the HVEMprotein. A preferred CRD1 domain is that set forth by residues 41-76 ofthe human HVEM protein sequence at Genbank accession no. AAB58354.1 (SEQID NO:6). See Montgomery et al., Cell, 87:427-436, 1996. Other preferredCRD1 domains are those having at least about 80%, 85%, 90% or 95%identity to the sequence set forth by residues 41-76 of the human HVEMprotein sequence at Genbank accession no. AAB58354.1 (SEQ ID NO:6).Another preferred CRD1 domain is that set forth by residues 39-80 of themurine HVEM protein sequence at Genbank accession no. AAQ08183.1 (SEQ IDNO:7). Other preferred CRD 1 domains are those having at least about80%, 85%, 90% or 95% identity to the sequence set forth by residues39-80 of the murine HVEM protein sequence at Genbank accession no.AAQ08183.1 (SEQ ID NO:7).

As used herein, the term “HVEM CRD1 domain peptide” refers to a peptidecorresponding in sequence to a region of the CRD1 domain of HVEM, whichpeptide can bind to the Ig domain of BTLA. An HVEM CRD1 domain peptideis capable of reducing the binding of the HVEM CRD1 domain to the BTLAIg domain, and is a BTLA-HVEM antagonist.

As used herein, the term “BTLA Ig domain” refers to the portion of aBTLA protein corresponding to the portion of BTLA that has been used toidentify the HVEM-BTLA interaction. In particular, the BTLA Ig domain,as used herein, comprises an immunogloublin domain. Further, as comparedto the BTLA sequence of C57BL/6 mouse, as found at Genbank accession no.NP 808252.1 (SEQ ID NO:1), the BTLA Ig domain corresponds to amino acids30-166. Further, as compared to the human BTLA sequence found at Genbankaccession no. AAP44003.1 (SEQ ID NO:2), the BTLA Ig domain correspondsto amino acids 31-149. A BTLA Ig domain binds to an HVEM CRD1 domain.Further, a fragment of a BTLA Ig domain binds to an HVEM CRD1 domain,and can be specifically bound by a preferred BTLA antibody disclosedherein. Some preferred BTLA Ig domains comprise a cysteine residuecorresponding to residue C85 of the murine BI/6 BTLA isoform (SEQ IDNO:1), which corresponds to residue C79 of the human BTLA isoform foundat Genbank accession no. AAP44003.1 (SEQ ID NO:2).

As used herein, the term “BTLA Ig domain peptide” refers to a peptidecorresponding in sequence to a region of the Ig domain of BTLA, whichpeptide can bind to the CRD1 domain of HVEM and is capable of reducingthe binding of the BTLA Ig domain to the HVEM CRD1 domain. Such peptidesare BTLA-HVEM antagonists.

As used herein, the term “HVEM blocking antibody” refers to an antibodythat specifically binds to HVEM and reduces binding of HVEM to BTLA.Preferred HVEM blocking antibodies bind to the CRD1 domain, morepreferably to a segment thereof that binds to the Ig domain of BTLA.

As used herein, the term “BTLA blocking antibody” refers to an antibodythat specifically binds to BTLA and reduces binding BTLA to HVEM.Preferred BTLA blocking antibodies bind to the Ig domain of BTLA,preferably to a segment thereof that binds to the HVEM CRD1 domain.

As used herein, the term “BTLA-HVEM antagonist” refers to a bioactiveagent capable of reducing BTLA activity in a cell having BTLA on itssurface. Preferred BTLA-HVEM antagonists are capable of reducing thebinding of HVEM on the surface of a cell to BTLA on the surface of thesame or a second cell. In some preferred embodiments, BTLA-HVEMantagonists are capable of binding to the BTLA Ig domain. Binding of aBTLA-HVEM antagonist to BTLA on the surface of a cell does not increaseBTLA activity in the cell.

As used herein, the term “BTLA-HVEM agonist” refers to a bioactive agentcapable of increasing BTLA activity in a cell having BTLA on itssurface, thereby mimicking the action of HVEM on BTLA. PreferredBTLA-HVEM agonists are capable of reducing the binding of HVEM on thesurface of a cell to BTLA on the surface of the same or a second cell.

Both HVEM and BTLA are synthesized and inserted into the plasma membraneas transmembrane proteins, and thereby expose respective extracellulardomains. The phrase “on the surface of a cell” in respect of BTLA orHVEM refers to non-soluble BTLA and HVEM protein localized at the plasmamembrane.

As used herein, the term “antagonistic HVEM antibody” refers to anantibody that specifically binds to HVEM and can reduce the ability ofHVEM to increase BTLA activity in a cell having BTLA on its surface.

As used herein, the term “antagonistic BTLA antibody” refers to anantibody that specifically binds to BTLA and can reduce the ability ofHVEM to increase BTLA activity in a cell having BTLA on its surface.Binding of an antagonistic BTLA antibody to BTLA on the surface of acell does not increase BTLA activity in the cell.

As used herein, the term “agonistic BTLA antibody” refers to an antibodythat specifically binds to BTLA, is capable of reducing the binding ofHVEM to BTLA, and increases BTLA activity in a cell having BTLA on itssurface.

By “BTLA activity” and variations thereof is meant intracellularsignaling and the effects thereof, caused by the binding of BTLA on thesurface of a cell by a BTLA agonist, e.g., HVEM on the surface of asecond cell; CMV UL144. BTLA activity includes but is not limited toinhibition of lymphocyte activation; phosphorylation of BTLAintracellular domain tyrosine residues, particularly those in the Grb2binding site, the immunoreceptor tyrosine-based inhibitory motif (ITIM),and/or the immunoreceptor tyrosine-based switch motif (ITSM); binding ofBTLA to SHP-1 and/or SHP-2; activation of SHP-1 and/or SHP-2; binding ofBTLA to Grb2; and binding of BTLA to p85 of PI3K.

By “modulating BTLA activity” is meant increasing or decreasing BTLAactivity, which includes completely decreasing BTLA activity such thatno BTLA activity is detectable.

As used herein, the term “lymphocyte activation” refers to the processesattendant B cell and T cell activation in primary or subsequent immuneresponses, which processes include but are not limited to cellproliferation, differentiation, migration, and survival, as well aseffector activities exhibited by B cells and T cells such as, but notlimited to, cytokine production, antibody production, Fas ligandproduction, chemokine production, granzyme production and release, andantigen presentation. Accordingly, as used herein, modulation oflymphocyte activation includes modulation of effector function, such asmodulation of the termination of effector function, etc. Numerous assaysare well known to the skilled artisan for detecting and/or monitoringsuch processes.

By “modulating lymphocyte activation” is meant increasing or decreasinglymphocyte activation, which includes decreasing lymphocyte activationsuch that no lymphocyte activation is detectable.

“Decreasing”, “reducing”, “inhibiting”, and grammatical equivalentsthereof are used interchangeably herein and refer to reductions inlevels of binding, activity, etc., which include reductions to levelsbeyond detection, including complete inhibition. Reduced binding can beeffected, for example, by competitive binding of an antagonist.

As used herein, the term “immune response” includes T and/or B cellresponses, i.e., cellular and/or humoral immune responses.

By “inhibiting tumor growth” is meant maintaining or reducing the tumorburden of an animal having an extant tumor, which includes eradicatingthe tumor. Even though the tumor burden is maintained or reduced, cancercell proliferation may be ongoing.

As used herein, “human antibodies” includes humanized antibodies.

It will be evident herein that the use of “BTLA” and “HVEM” refers toBTLA protein and HVEM protein in many instances.

In some embodiments herein, CRD1 domains, and BTLA Ig domains areidentified by their percent identity to a particular CRD1 or BTLAsequence. As is known in the art, a number of different programs can beused to identify whether a protein or nucleic acid has sequence identityor similarity to a known sequence. For a detailed discussion, see D.Mount, Bioinformatics, Cold Spring Harbor Press, Cold Spring Harbor,N.Y., 2001, ISBN 0-87969-608-7. Sequence identity and/or similarity isdetermined using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith &Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identityalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, PNAS USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Drive, Madison, Wis.), theBest Fit sequence program described by Devereux et al., Nucl. Acid Res.12:387-395 (1984), preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST programis the WU-BLAST-2 program which was obtained from Altschul et al.,Methods in Enzymology, 266: 460-480 (1996)]. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions; charges gap lengths of k a cost of 10+k;Xu set to 16, and Xg set to 40 for database search stage and to 67 forthe output stage of the algorithms. Gapped alignments are triggered by ascore corresponding to ˜22 bits. A percent amino acid sequence identityvalue is determined by the number of matching identical residues dividedby the total number of residues of the longer sequence in the alignedregion. The longer sequence is the one having the most actual residuesin the aligned region (gaps introduced by WU-Blast-2 to maximize thealignment score are ignored).

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the protein sequences set forth in the figures,it is understood that in one embodiment, the percentage of sequenceidentity will be determined based on the number of identical amino acidsin relation to the total number of amino acids. Thus, for example, thepercent sequence identity of sequences shorter than those shown in thefigures will be determined using the number of amino acids in theshorter sequence, in one embodiment. In percent identity calculationsrelative weight is not assigned to various manifestations of sequencevariation, such as, insertions, deletions, substitutions, etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of 0,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the shortersequence in the aligned region and multiplying by 100. The longersequence is the one having the most actual residues in the alignedregion.

In a similar manner, percent (%) nucleic acid sequence identity isdefined as the percentage of nucleotide residues in a candidate sequencethat are identical with the nucleotide residues in the B7x nucleic acidset forth in FIG. 2 or 4, or a BTLA nucleic acid sequence encoding aBTLA amino acid sequence set forth in FIG. 19. A preferred methodutilizes the BLASTN module of WU-BLAST-2 set to the default parameters,with overlap span and overlap fraction set to 1 and 0.125, respectively.

BTLA Antibodies and HVEM Antibodies

Antibody Structure

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. Seegenerally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). The variable regions of each light/heavy chain pair form theantibody binding site.

Thus, an intact IgG antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J. Immunol. 148:1547-1553 (1992). In addition, bispecificantibodies may be formed as “diabodies” (Holliger et al. “‘Diabodies’:small bivalent and bispecific antibody fragments” PNAS USA 90:6444-6448(1993)) or “Janusins” (Traunecker et al. “Bispecific single chainmolecules (Janusins) target cytotoxic lymphocytes on HIV infected cells”EMBO J. 10:3655-3659 (1991) and Traunecker et al. “Janusin: newmolecular design for bispecific reagents” Int J Cancer Suppl 7:51-52(1992)). Production of bispecific antibodies can be a relatively laborintensive process compared with production of conventional antibodiesand yields and degree of purity are generally lower for bispecificantibodies. Bispecific antibodies do not exist in the form of fragmentshaving a single binding site (e.g., Fab, Fab′, and Fv).

Human Antibodies and Humanization of Antibodies

Human antibodies avoid certain of the problems associated withantibodies that possess murine or rat variable and/or constant regions.The presence of such murine or rat derived proteins can lead to therapid clearance of the antibodies or can lead to the generation of animmune response against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, it has been postulatedthat one can develop humanized antibodies or generate fully humanantibodies through the introduction of human antibody function into arodent so that the rodent would produce antibodies having fully humansequences.

Human Antibodies

Introduction of human immunoglobulin (Ig) loci into mice in which theendogenous Ig genes have been inactivated provides an ideal source forproduction of fully human monoclonal antibodies (Mabs). Fully humanantibodies are expected to minimize the immunogenic and allergicresponses intrinsic to mouse or mouse-derivatized Mabs and thus toincrease the efficacy and safety of the administered antibodies. The useof fully human antibodies can be expected to provide a substantialadvantage in the treatment of chronic and recurring human diseases, suchas cancer, which may require repeated antibody administrations.

Mouse strains have been engineered with large fragments of the human Igloci and to produce human antibodies in the absence of mouse antibodies.

See Green et al. Nature Genetics 7:13-21 (1994). The XenoMouse™ strainswere engineered with yeast artificial chromosomes (YACs) containing 245kb and 190 kb-sized germline configuration fragments of the human heavychain locus and kappa light chain locus, respectively, which containedcore variable and constant region sequences. Further reported workinvolved the introduction of greater than approximately 80% of the humanantibody repertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and kappalight chain loci, respectively, to produce XenoMouse™ mice. See Mendezet al. Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp.Med. 188:483-495 (1998), the disclosures of which are herebyincorporated by reference.

Such approaches are further discussed and delineated in European PatentNo., EP 0 463 151 B1, grant published Jun. 12, 1996, InternationalPatent Application No., WO 94/02602, published Feb. 3, 1994,International Patent Application No., WO 96/34096, published Oct. 31,1996, and WO 98/24893, published Jun. 11, 1998. The disclosures of eachof the above-cited patents, applications, and references are herebyincorporated by reference in their entirety.

In an alternative approach, others have utilized a “minilocus” approach.In the minilocus approach, an exogenous Ig locus is mimicked through theinclusion of pieces (individual genes) from the Ig locus. Thus, one ormore VH genes, one or more DH genes, one or more JH genes, a μ constantregion, and a second constant region (preferably a gamma constantregion) are formed into a construct for insertion into an animal. Thisapproach is described in U.S. Pat. No. 5,545,807 to Surani et al. andU.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016,5,770,429, 5,789,650, and 5,814,318 each to Lonberg and Kay, U.S. Pat.No. 5,591,669 to Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205,5,721,367, 5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 toChoi and Dunn. See also European Patent No. 0 546 073 B1, InternationalPatent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO97/13852, and WO 98/24884, the disclosures of which are herebyincorporated by reference in their entirety. See further Taylor et al.,Nucleic Acids Research 20:6287-6295 (1992); Chen et al. InternationalImmunology 5:647-656 (1993); Tuaillon et al., Proc. Natl. Acad. Sci. USA90:3720-3724 (1993); Choi et al., Nature Genetics 4:117-123 (1993);Lonberg et al., Nature 368:856-859 (1994); Taylor et al., InternationalImmunology 6:579-59.1 (1994); Tuaillon et al., J. Immunol. 154:6453-6465(1995); Fishwild et al., Nature Biotech. 14:845-851 (1996); thedisclosures of which are hereby incorporated by reference in theirentirety.

BTLA and HVEM proteins and fragments thereof, HVEM CRD1 domain peptides,BTLA Ig domain peptides, BTLA fusion proteins, and HVEM fusion proteinsmay be used to generate BTLA antibodies and HVEM antibodies of thepresent invention.

The term “antibody” as used herein includes both monoclonal andpolyclonal antibodies as well as antibody fragments, as are known in theart, including Fab, F(ab)₂, single chain antibodies (Fv for example),chimeric antibodies, humanized antibodies, etc., either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA technologies. Antibody fragments include those portionsof the antibody that bind to an HVEM CRD1 domain or a BTLA Ig domain.

Preferably, when a BTLA or HVEM protein fragment is to be used as animmunogen to generate antibodies, the fragment must share at least oneepitope or determinant with the full length protein, particularly in anHVEM CRD1 domain or a BTLA Ig domain. By epitope or determinant hereinis meant a portion of a protein which will generate and/or bind anantibody. Thus, in most instances, antibodies made to a smaller ortruncated BTLA or HVEM protein will be able to bind to the correspondingfull length protein. In a preferred embodiment, the epitope is unique;that is, antibodies generated to a unique epitope show little or nocross-reactivity.

In one embodiment, the invention provides antagonistic BTLA antibodiesthat are capable of reducing, including eliminating, one or morebiological functions of the BTLA protein expressed at the surface of acell. That is, the addition of an antagonistic BTLA antibody(polyclonal, or preferably monoclonal) to a cell expressing BTLA at itssurface can reduce or eliminate at least one BTLA activity. BTLAactivity includes but is not limited to the inhibition of lymphocyteactivation; phosphorylation of tyrosine residues in the Grb2 bindingsite, the ITIM, or the ITSM; binding to SHP-1 and/or SHP-2; andactivation of SHP-1 and/or SHP-2. The reduction of BTLA activity isobserved in the presence of BTLA agonist (eg. HVEM on the surface of asecond cell) which stimulates BTLA activity in the absence of anantagonistic BTLA antibody. In a preferred embodiment, such anantagonistic BTLA antibody interferes with the binding of HVEM on thesurface of one cell to BTLA on the surface of a second cell.

Generally, at least a 25% decrease in BTLA activity is preferred, withat least about 50% being particularly preferred and about a 95-100%decrease being especially preferred.

Such antagonistic BTLA antibodies are sometimes referred to herein asfunction-blocking antibodies. Such antibodies have the ability toincrease B and T lymphocyte activation by decreasing BTLA activity inlymphocytes. Further, such antibodies have the ability to modulateimmunoglobulin production by B cells expressing BTLA, and moreparticularly, to increase immunoglobulin production.

In an alternative embodiment, the invention provides agonistic BTLAantibodies that increase or potentiate one or more biological functionsof the BTLA protein expressed at the surface of a cell. That is, theaddition of an agonistic BTLA antibody (polyclonal, or preferablymonoclonal) to a cell expressing BTLA at its surface will increase orpotentiate at least one BTLA activity. BTLA activity includes but is notlimited to the inhibition of lymphocyte activation; phosphorylation oftyrosine residues in the Grb2 binding site, the ITIM, or the ITSM;binding to SHP-1 and/or SHP-2; and activation of SHP-1 and/or SHP-2.

In a preferred embodiment, the agonistic BTLA antibodies arefunction-activating antibodies. Such antibodies have the ability todecrease B and T lymphocyte activation by increasing BTLA activity inlymphocytes. Further, such antibodies have the ability to modulateimmunoglobulin production by B cells expressing BTLA, and moreparticularly, to decrease immunoglobulin production.

A BTLA antibody of the invention specifically binds to the BTLA Igdomain of a BTLA protein. By “specifically bind” herein is meant thatthe antibodies bind to the protein with a binding constant in the rangeof at least 10⁻⁴-10⁻⁶ M⁻¹, with a preferred range being 10⁻⁷-10⁻⁹ M⁻¹.

The BTLA proteins bound by BTLA antibodies may be human BTLA proteins,murine BTLA proteins, or other, preferably mammalian, BTLA proteins. Ina preferred embodiment, the BTLA protein is a human BTLA protein.

The murine BTLA gene is polymorphic, and variations in sequence withinthe Ig domain that binds to murine HVEM are described herein in thefigures. Despite their sequence variation, the murine BTLA Ig domainsare each capable of binding to murine HVEM, and a number of BTLAblocking antibodies are capable of binding to multiple isoforms ofmurine BTLA.

The human BTLA gene is also polymorphic, as disclosed in U.S.application Ser. No. 10/600,997, expressly incorporated herein in itsentirety by reference. As disclosed herein, human HVEM is capable ofbinding to human BTLA. It is within the skill of the artisan todetermine if alternative alleles of human BTLA are capable of binding toHVEM. As used herein, the term “BTLA” includes any human isoform of BTLAthat is capable of binding to HVEM.

In a preferred embodiment, the present invention provides monoclonalBTLA antibodies that specifically bind to murine and/or human BTLAproteins.

In one embodiment, the invention provides antagonistic HVEM antibodiesthat are capable of reducing, including eliminating, the ability of HVEMprotein when expressed at the surface of a cell to increase BTLAactivity in a second cell expressing BTLA at its surface. BTLA activityincludes but is not limited to the inhibition of lymphocyte activation;phosphorylation of tyrosine residues in the Grb2 binding site, the ITIM,or the ITSM; binding to SHP-1 and/or SHP-2; and activation of SHP-1and/or SHP-2. In a preferred embodiment, such an antagonistic HVEMantibody interferes with the binding of HVEM on the surface of one cellto BTLA on the surface of a second cell.

Generally, at least a 25% decrease in activity is preferred, with atleast about 50% being particularly preferred and about a 95-100%decrease being especially preferred.

Such antibodies have the ability to increase B and T lymphocyteactivation by decreasing BTLA activity in lymphocytes. Further, suchantibodies have the ability to modulate immunoglobulin production by Bcells expressing BTLA, and more particularly, to increase immunoglobulinproduction.

The HVEM antibodies of the invention specifically bind to HVEM CRD1domains. By “specifically bind” herein is meant that the antibodies bindto the protein with a binding constant in the range of at least10⁻⁴-10⁻⁶ M⁻¹, with a preferred range being 10⁻⁷-10⁻⁹ M⁻¹.

The HVEM proteins bound by HVEM antibodies may be human HVEM proteins,murine HVEM proteins, or other, preferably mammalian, HVEM proteins.

HVEM protein sequences and encoding nucleic acid sequences are wellknown in the art. For example, see Montgomery et al., Cell, 87: 427-436,1996; Kwon et al., Journal of Biological Chemistry, 272:14272-14276,1997; Hsu et al., Journal of Biological Chemistry 272:13471-13474, 1997.

The term “antibody”, as used herein, includes immunoglobulin moleculescomprised of four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as HCVRor VH) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CHI, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The phrase “complementary determining region” (CDR) includesthe region of an antibody molecule which comprises the antigen bindingsite.

The antibody may be an IgG such as IgG1, IgG2, IgG3 or IgG4; or IgM,IgA, IgE or IgD isotype. The constant domain of the antibody heavy chainmay be selected depending upon the effector function desired. The lightchain constant domain may be a kappa or lambda constant domain.

The term “antibody” as used herein also encompasses antibody fragments,and in particular, fragments that retain the ability to specificallybind to an antigen. It has been shown that the antigen-binding functionof an antibody can be performed by fragments of a full-length antibody.Examples of such binding fragments include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CHI domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antibody.” Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger, P., et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al.(1994) Structure 2:1121-1123).

Still further, an antibody or fragment thereof may be part of a largerimmunoadhesion molecule, formed by covalent or noncovalent associationof the antibody or antibody portion with one or more other proteins orpeptides. Examples of such immunoadhesion molecules include use of thestreptavidin core region to make a tetrameric scFv molecule (Kipriyanov,S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and useof a cysteine residue, a marker peptide and a C-terminal polyhistidinetag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M.,et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such asFab and F(ab′)₂ fragments, can be prepared from whole antibodies usingconventional techniques, such as papain or pepsin digestion,respectively, of whole antibodies. Moreover, antibodies, antibodyfragments and immunoadhesion molecules can be obtained using standardrecombinant DNA techniques, as described herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof, e.g. humanized, chimeric, etc.Preferably, antibodies of the invention bind specifically orsubstantially specifically to an HVEM CRD1 domain or a BTLA Ig domain.The terms “monoclonal antibodies” and “monoclonal antibody composition”,as used herein, refer to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of an antigen, whereas the term “polyclonalantibodies” and “polyclonal antibody composition” refer to a populationof antibody molecules that contain multiple species of antigen bindingsites capable of interacting with a particular antigen. A monoclonalantibody composition typically displays a single binding affinity for aparticular antigen with which it immunoreacts.

The antibodies described herein may be humanized antibodies, e.g.,antibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germlineimmunoglobulin sequences. The humanized antibodies of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs. Such humanized antibodies may also include antibodies in which CDRsequences derived from the germline of another mammalian species, suchas a mouse, have been grafted onto human framework sequences.

BTLA-Binding Domain Peptides and HVEM-Binding Domain Peptides

As used herein, peptide refers to at least two covalently attached aminoacids, which includes proteins, polypeptides, and oligopeptides. Theprotein may be made up of naturally occurring amino acids and peptidebonds, or synthetic peptidomimetic structures. Thus, “amino acid” or“peptide residue” as used herein means both naturally occurring andsynthetic amino acids. For example, homo-phenylalanine, citrulline, andnorleucine are considered amino acids for the purposes of the invention.“Amino acids” also includes imino residues such as proline andhydroxyproline. The side chains may be either the D- or L-configuration,or combinations thereof. Although the bond between each amino acid istypically an amide or peptide bond, it is to be understood that peptidealso includes analogs of peptides in which one or more peptide linkagesare replaced with other than an amide or peptide linkage, such as asubstituted amide linkage, an isostere of an amide linkage, or a peptideor amide mimetic linkage (See™, for example, Spatola, “Peptide BackboneModifications,” in Chemistry and Biochemistry of Amino Acids Peptidesand Proteins, Weinstein, Ed., Marcel Dekker, New York (1983); Son etal., J. Med. Chem. 36:3039-3049 (1993); and Ripka and Rich, Curr. Opin.Chem. Biol. 2:441-452 (1998)).

Typically, peptides will generally be less than about 100 amino acids,less that about 50 amino acids, or less than about 20 amino acids.

A peptide herein is typically an isolated or purified peptide. As usedherein, a peptide is said to be “isolated” or “purified” when it issubstantially free of cellular material or free of chemical precursorsor other chemicals. The peptides of the present invention can bepurified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The phrase“substantially free of chemical precursors or other chemicals” includespreparations of the peptide in which it is separated from chemicalprecursors or other chemicals that are involved in its synthesis.Preparations of a peptide are substantially free of precursors inpreparation having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

The peptides of this invention can be made by chemical synthesis methodswhich are well known to the ordinarily skilled artisan. See, forexample, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide,ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Peptidescan be synthesized using the automated Merrifield techniques of solidphase synthesis with the αNH2 protected by either t-Boc or Fmocchemistry using side chain protected amino acids on, for example, anApplied Biosystems Peptide Synthesizer Model 430A or 431.

After complete assembly of the desired peptide, the resin is treatedaccording to standard procedures to cleave the peptide from the resinand deblock the functional groups on the amino acid side chains. Thefree peptide is purified, for example by HPLC, and characterizedbiochemically, for example, by amino acid analysis, mass spectrometry,and/or by sequencing. Purification and characterization methods forpeptides are well known to those of ordinary skill in the art.

Longer synthetic peptides can be synthesized by well-known recombinantDNA techniques. Many standard manuals on molecular cloning technologyprovide detailed protocols to produce the peptides of the invention byexpression of recombinant DNA and RNA. To construct a gene encoding apeptide of this invention, the amino acid sequence is reverse translatedinto a nucleic acid sequence, preferably using optimized codon usage forthe organism in which the gene will be expressed. Next, a gene encodingthe peptide is made, typically by synthesizing overlappingoligonucleotides which encode the peptide and necessary regulatoryelements. The synthetic gene is assembled and inserted into the desiredexpression vector. Nucleic acids which comprise sequences that encodethe peptides of this invention are also provided. The synthetic gene isinserted into a suitable cloning vector and recombinants are obtainedand characterized. The peptide is then expressed under conditionsappropriate for the selected expression system and host. The peptide ispurified and characterized by standard methods.

Fusion Proteins

Variant polypeptides of the present invention may also be fused toanother, heterologous polypeptide or amino acid sequence to form achimera. In some embodiments, fusion proteins comprise fusion partnerscomprising labels (e.g. autofluorescent proteins, survival and/orselection proteins), stability and/or purification sequences, toxins, orany other protein sequences of use. Additional fusion partners aredescribed below. In some instances, the fusion partner is not a protein.

In another embodiment, a polypeptide of the invention is fused withhuman serum albumin to improve pharmacokinetics.

In a further embodiment, a polypeptide of the invention is fused to acytotoxic agent. In this method, the polypeptide of the invention actsto target the cytotoxic agent to cells, resulting in a reduction in thenumber of afflicted cells. Cytotoxic agents include, but are not limitedto, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain,curcin, crotin, phenomycin, enomycin and the like, as well asradiochemicals.

Peptide Tags

Various tag polypeptides and their respective antibodies are well knownin the art. Epitope tags may be placed at the amino- orcarboxyl-terminus of a polypeptide of the invention to enable antibodydetection. Also, the epitope tag enables a polypeptide of the inventionto be readily purified by affinity purification. Examples of peptidetags include, but are not limited to, poly-histidine (poly-His) orpoly-histidine-glycine (poly-His-Gly) tags; the flu HA tag polypeptide[Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)]; the c-myc tag [Evanet al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; the HerpesSimplex virus glycoprotein D (gD) tag [Paborsky et al., ProteinEngineering, 3(6):547-553 (1990)], the Flag-peptide [Hopp et al.,BioTechnology 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science 255:192-194 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem. 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. U.S.A.87:6393-6397 (1990)].

Labels

In one embodiment, a polypeptide of the invention is modified by theaddition of one or more labels. For example, labels that may be used arewell known in the art and include but are not limited to biotin, tag andfluorescent labels (e.g. fluorescein). These labels may be used invarious assays as are also well known in the art to achievecharacterization.

Additional BTLA-HVEM Agonists and BTLA-HVEM Antagonists

It will be appreciated that additional bioactive agents may be screenedfor BTLA-HVEM antagonistic activity and BTLA-HVEM agonistic activity. Ina preferred embodiment, candidate bioactive agents are screened fortheir ability to reduce binding of BTLA to HVEM. In another preferredembodiment, candidate bioactive agents are screened for their ability toreduce BTLA activation by HVEM.

The assays preferably utilize human BTLA and human HVEM proteins,although other BTLA and HVEM proteins may also be used.

In a preferred embodiment, the methods comprise combining an Ig domainof a BTLA protein, or an HVEM binding portion thereof, with a candidatebioactive agent, and determining the binding of the candidate agent tothe BTLA domain. In another preferred embodiment, the methods involvecombining an HVEM CRD1 domain, or a BTLA binding portion thereof, with acandidate agent, and determining the binding of the candidate agent tothe HVEM domain.

The term “candidate bioactive agent” as used herein describes anymolecule, e.g., protein, small organic molecule, carbohydrates(including polysaccharides), polynucleotide, lipids, etc. Generally aplurality of assay mixtures are run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e., at zero concentration or below the level ofdetection. In addition, positive controls, i.e. the use of agents knownto bind BTLA or HVEM may be used.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons,more preferably between 100 and 2000, more preferably between about 100and about 1250, more preferably between about 100 and about 1000, morepreferably between about 100 and about 750, more preferably betweenabout 200 and about 500 daltons. Candidate agents comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Particularly preferred arepeptides, e.g., peptidomimetics. Peptidomimetics can be made asdescribed, e.g., in WO 98/56401.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In a preferred embodiment, the candidate bioactive agents are organicchemical moieties or small molecule chemical compositions, a widevariety of which are available in the art.

Additional Therapeutic Agents

In a further embodiment, the bioactive agents disclosed herein,including BTLA-HVEM agonists and BTLA-HVEM antagonists, includingantagonistic BTLA antibodies and agonistic BTLA antibodies, may beadvantageously combined with one or more additional therapeutic agents.

In one aspect, the BTLA-HVEM antagonists described herein can beadministered in combination with additional immune response stimulatingagents such as, e.g., cytokines as well as various antigens and vaccinepreparations including tumor antigens and tumor vaccines. In preferredembodiments, such cytokines stimulate antigen presenting cells, e.g.,GM-CSF, M-CSF, G-CSF, IL-3, IL-12, etc. Additional proteins and/orcytokines known to enhance T cell proliferation and secretion, such asIL-2, IL-2, B7, anti-CD3 and anti-CD28 can be employed simultaneously orsequentially with the BTLA-HVEM antagonists to augment the immuneresponse. The subject therapy may also be combined with the transfectionor transduction of tumor cells with genes encoding for various cytokinesor cell surface receptors, as is known in the art. See, e.g. Ogasawaraet al. (1993) Cancer Res. 53:3561-8 and Townsend et al., (1993) Science259:368-370.

In another aspect, the BTLA-HVEM agonists described herein can beadministered in combination with immunosuppressive agents, e.g.,antibodies against other lymphocyte surface markers (e.g., CD40) oragainst cytokines, other fusion proteins, e.g., CTLA4Ig, or otherimmunosuppressive drugs (e.g., cyclosporin A, FK506-like compounds,rapamycin compounds, or steroids).

It is further contemplated that methods using BTLA-HVEM antagonists andBTLA-HVEM agonists may be synergistically combined with immunotherapiesbased on modulation of other positive and negative costimulatorypathways, and with CTLA-4 modulation in particular. For example,BTLA-HVEM antagonists may be advantageously combined with CTLA-4blocking agents as described in U.S. Pat. Nos. 5,855,887; 5,811,097; and6,051,227, and International Publication WO 00/32231. Such CTLA-4blocking agents inhibit T cell down-regulation mediated by CTLA-4interaction with B7 family members B71 and B72 expressed on lymphoid anddendritic cells. Similarly, BTLA-HVEM agonists may be advantageouslycombined with CTLA-4 mimicking agents such as CTLA-4Ig, which hasalready found clinical use as an immunosuppressive agent.

As used herein the term “rapamycin compound” includes the neutraltricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs,and other macrolide compounds which are thought to have the samemechanism of action as rapamycin (e.g., inhibition of cytokinefunction). The language “rapamycin compounds” includes compounds withstructural similarity to rapamycin, e.g., compounds with a similarmacrocyclic structure, which have been modified to enhance theirtherapeutic effectiveness. Exemplary Rapamycin compounds suitable foruse in the invention, as well as other methods in which Rapamycin hasbeen administered are known in the art (See, e.g. WO 95/22972, WO95/16691, WO 95/04738, U.S. Pat. Nos. 6,015,809; 5,989,591; U.S. Pat.Nos. 5,567,709; 5,559,112; 5,530,006; 5,484,790; 5,385,908; 5,202,332;5,162,333; 5,780,462; 5,120,727).

The language “FK506-like compounds” includes FK506, and FK506derivatives and analogs, e.g., compounds with structural similarity toFK506, e.g., compounds with a similar macrocyclic structure which havebeen modified to enhance their therapeutic effectiveness. Examples ofFK506 like compounds include, for example, those described in WO00/01385. Preferably, the language “rapamycin compound” as used hereindoes not include FK506-like compounds.

Administration of Therapeutic Compositions

The bioactive agents of the present invention are administered tosubjects in a biologically compatible form suitable for pharmaceuticaladministration in vivo. By “biologically compatible form suitable foradministration in vivo” is meant a form of the agent to be administeredin which any toxic effects are outweighed by the therapeutic effects ofthe agent. The term subject is intended to include living organisms inwhich an immune response can be elicited, e.g., mammals. Examples ofsubjects include humans, dogs, cats, mice, rats, and transgenic speciesthereof. Administration of a bioactive agent as described herein can bein any pharmacological form, including a therapeutically active amountof a BTLA antibody, optionally in combination with an additionaltherapeutic agent as described herein, and a pharmaceutically acceptablecarrier. Administration of a therapeutically effective amount of thetherapeutic compositions of the present invention is defined as anamount effective, at dosages and for periods of time necessary toachieve the desired therapeutic result. For example, a therapeuticallyactive amount of a BTLA antibody may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the antibody to elicit a desired response in the individual.A dosage regime may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation.

The bioactive agent (e.g., BTLA antibody) may be administered in aconvenient manner such as by injection (subcutaneous, intravenous,etc.), oral administration, inhalation, transdermal application, orrectal administration. Depending on the route of administration, thebioactive agent may be coated in a material to protect the compound fromthe action of enzymes, acids and other natural conditions which mayinactivate the compound.

To administer a bioactive agent comprising a protein, e.g. a BTLAantibody, by other than parenteral administration, it may be necessaryto coat the protein with, or co-administer the protein with, a materialto prevent its inactivation. A bioactive agent such as a BTLA antibodymay be administered to an individual in an appropriate carrier, diluentor adjuvant, co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. Pharmaceutically acceptable diluents includesaline and aqueous buffer solutions. Adjuvant is used in its broadestsense and includes any immune stimulating compound such as interferon.Exemplary adjuvants include alum, resorcinols, non-ionic surfactantssuch as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.Enzyme inhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water emulsions as well as conventional liposomes(Strejan et al., (1984) J. Neuroimmunol 7:27).

The bioactive agent may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

In one embodiment, a pharmaceutical composition suitable for injectableuse includes sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. In all cases, thecomposition will preferably be sterile and fluid to the extent that easysyringability exists. It will preferably be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating one ormore bioactive agents, together or separately with additional immuneresponse stimulating agents or immunosupressants, in the required amountin an appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the bioactive agentinto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

When a bioactive agent comprising a peptide is suitably protected, asdescribed above, the protein may be orally administered, for example,with an inert diluent or an assimilable edible carrier. As used herein“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the therapeuticcompositions is contemplated. Supplementary bioactive agents can also beincorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of bioactive agent calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the bioactive agent(s) and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an agent for the treatment of sensitivity inindividuals.

The specific dose can be readily calculated by one of ordinary skill inthe art, e.g., according to the approximate body weight or body surfacearea of the patient or the volume of body space to be occupied. The dosewill also be calculated dependent upon the particular route ofadministration selected. Further refinement of the calculationsnecessary to determine the appropriate dosage for treatment is routinelymade by those of ordinary skill in the art. Such calculations can bemade without undue experimentation by one skilled in the art in light ofthe activity disclosed herein in assay preparations of target cells.Exact dosages are determined in conjunction with standard dose-responsestudies. It will be understood that the amount of the compositionactually administered will be determined by a practitioner, in the lightof the relevant circumstances including the condition or conditions tobe treated, the choice of composition to be administered, the age,weight, and response of the individual patient, the severity of thepatient's symptoms, and the chosen route of administration.

The toxicity and therapeutic efficacy of the bioactive agents describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD50 (thedose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Compounds which exhibit largetherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch agents lies preferably within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any agent used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test agent which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In one embodiment of the present invention a therapeutically effectiveamount of an antibody to BTLA or HVEM is administered to a subject. Asdefined herein, a therapeutically effective amount of antibody (i.e., aneffective dosage) ranges from about 0.001 to 50 mg/kg body weight,preferably about 0.01 to 40 mg/kg body weight, more preferably about 0.1to 30 mg/kg body weight, about 1 to 25 mg/kg, 2 to 20 mg/kg, 5 to 15mg/kg, or 7 to 10 mg/kg body weight. The optimal dose of the antibodygiven may even vary in the same patient depending upon the time at whichit is administered.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of an antibody can include a single treatment or,preferably, can include a series of treatments. In a preferred example,a subject is treated with antibody in the range of between about 0.1 to20 mg/kg body weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody used for treatmentmay increase or decrease over the course of a particular treatment.Changes in dosage may result from the results of assays designed tomonitor transplant status (e.g., whether rejection or an immune responsein the subject has occurred) as known in the art or as described herein.

In one embodiment, a pharmaceutical composition for injection could bemade up to contain 1 ml sterile buffered water, and 1 to 50 mg ofantibody. A typical composition for intravenous infusion could be madeup to contain 250 ml of sterile Ringer's solution, and 150 mg ofantibody. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in, for example, Remington's PharmaceuticalScience, 15th ed., Mack Publishing Company, Easton, Pa. (1980), which isincorporated herein by reference. The compositions comprising thepresent antibodies can be administered for prophylactic and/ortherapeutic treatments. In therapeutic application, compositions can beadministered to a patient already suffering from a disease, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the clinical situation and the general state of thepatient's own immune system. For example, doses for preventingtransplant rejection may be lower than those given if the patientpresents with clinical symptoms of rejection. Single or multipleadministrations of the compositions can be carried out with dose levelsand pattern being selected by the treating physician. In any event, thepharmaceutical formulations should provide a quantity of the bioactiveagents described herein sufficient to effectively treat the patient.

Dose administration can be repeated depending upon the pharmacokineticparameters of the dosage formulation and the route of administrationused. It is also provided that certain protocols may allow for one ormore agents describe herein to be administered orally. Such formulationsare preferably encapsulated and formulated with suitable carriers insolid dosage forms. Some examples of suitable carriers, excipients, anddiluents include lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, calcium silicate,microcrystalline cellulose, olyvinylpyrrolidone, cellulose, gelatin,syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc,magnesium, stearate, water, mineral oil, and the like. The formulationscan additionally include lubricating agents, wetting agents, emulsifyingand suspending agents, preserving agents, sweetening agents or flavoringagents. The compositions may be formulated so as to provide rapid,sustained, or delayed release of the active ingredients afteradministration to the patient by employing procedures well known in theart. The formulations can also contain substances that diminishproteolytic degradation and/or substances which promote absorption suchas, for example, surface active agents.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. Kits forpractice of the instant invention are also provided. For example, such akit comprises a bioactive agent such as, e.g., an antibody reactive withBTLA or HVEM, together with a means for administering the antibodyconjugate, e.g., one or more syringes. The kit can come packaged withinstructions for use.

Modulation of Immune Responses

The present invention provides methods for modulating lymphocyteactivity and immune responses to antigens using BTLA-HVEM antagonistsand BTLA-HVEM agonists described herein. The methods are useful formodulating the activity of, for example, naïve T cells, CD8+ T_(c)cells, CD4+ cells, T_(H)1 cells, and B cells.

BTLA-HVEM antagonists are used alone or in combination with othertherapeutic agents to reduce the negative costimulatory signals emittedby BTLA, and to reduce the suppression and/or attenuation of lymphocyteactivity mediated by BTLA signaling.

BTLA-HVEM agonists are used are used alone or in combination with othertherapeutic agents to increase negative costimulatory signals emitted byBTLA, thereby increasing the suppression and/or attenuation oflymphocyte activity mediated by BTLA signaling.

In a preferred embodiment, the methods comprise contacting a lymphocyteexpressing BTLA on its surface, or a second cell expressing HVEM on itssurface, or both, with a BTLA-HVEM antagonist, wherein the lymphocyteand second cell are able to contact each other such that BTLA on thelymphocyte can bind to HVEM on the second cell, and wherein theBTLA-HVEM antagonist reduces the activation of BTLA on the lymphocyte byHVEM on the second cell.

In another preferred embodiment, the methods comprise contacting alymphocyte expressing BTLA on its surface with a BTLA-HVEM agonist, suchthat the BTLA-HVEM agonist increases BTLA activity in the lymphocyte.

Antigens

In one aspect, the invention provides methods for modulating an immuneresponse to antigen. Such antigens can be, for example, tumor-associatedantigens, pathogen antigens, and autoantigens (self antigens). Antigenicstimulation may be a result of ongoing malignancy or infection, and/ormay be a result of exposure to antigens delivered by vaccine.

A wide variety of antigens may find use in the invention. In particular,the adjuvant compositions provided herein may be advantageously combinedwith antigenic stimulation from tumor-associated antigens or pathogenantigens to increase lymphocyte activity against the corresponding tumoror pathogen. Generally, suitable antigens may be derived from proteins,peptides, polypeptides, lipids, glycolipids, carbohydrates and DNA foundin the subject tumor or pathogen.

Tumor-associated antigens finding utility herein include both mutatedand non-mutated molecules which may be indicative of a single tumortype, shared among several types of tumors, and/or exclusively expressedor over-expressed in tumor cells in comparison with normal cells. Inaddition to proteins and glycoproteins, tumor-specific patterns ofexpression of carbohydrates, gangliosides, glycolipids and mucins havealso been documented.

Exemplary tumor-associated antigens for use in the subject cancervaccines include protein products of oncogenes, tumor suppressor genesand other genes with mutations or rearrangements unique to tumor cells,reactivated embryonic gene products, oncofetal antigens, tissue-specific(but not tumor-specific) differentiation antigens, growth factorreceptors, cell surface carbohydrate residues, foreign viral proteinsand a number of other self proteins.

Specific embodiments of tumor-associated antigens include, e.g., mutatedantigens such as the protein products of the Ras p21 protooncogenes,tumor suppressor, p53 and HER-2/neu and BCR-abl oncogenes, as well asCDK4, MUM1, Caspase 8, and Beta catenin; overexpressed antigens such asgalectin 4, galectin 9, carbonic anhydrase, Aldolase A, PRAME, Her2/neu,ErbB-2 and KSA, oncofetal antigens such as alpha fetoprotein (AFP),human chorionic gonadotropin (hCG); self antigens such ascarcinoembryonic antigen (CEA) and melanocyte differentiation antigenssuch as Mart 1/Melan A, gp100, gp75, Tyrosinase, TRP1 and TRP2; prostateassociated antigens such as PSA, PAP, PSMA, PSM-P1 and PSM-P2;reactivated embryonic gene products such as MAGE 1, MAGE 3, MAGE 4, GAGE1, GAGE 2, BAGE, RAGE, and other cancer testis antigens such as NY-ESO1,SSX2 and SCP1; mucins such as Muc-1 and Muc-2; gangliosides such as GM2,GD2 and GD3, neutral glycolipids and glycoproteins such as Lewis (y) andglobo-H; and glycoproteins such as Tn, Thompson-Freidenreich antigen(TF) and sTn. Also included as tumor-associated antigens herein arewhole cell and tumor cell lysates as well as immunogenic portionsthereof, as well as immunoglobulin idiotypes expressed on monoclonalproliferations of B lymphocytes for use against B cell lymphomas.

Tumor-associated antigens and their respective tumor cell targetsinclude, e.g., cytokeratins, particularly cytokeratin 8, 18 and 19, asantigens for carcinoma. Epithelial membrane antigen (EMA), humanembryonic antigen (HEA-125), human milk fat globules, MBr1, MBr8,Ber-EP4, 17-1A, C26 and T16 are also known carcinoma antigens. Desminand muscle-specific actin are antigens of myogenic sarcomas. Placentalalkaline phosphatase, beta-human chorionic gonadotropin, andalpha-fetoprotein are antigens of trophoblastic and germ cell tumors.Prostate specific antigen is an antigen of prostatic carcinomas,carcinoembryonic antigen of colon adenocarcinomas. HMB-45 is an antigenof melanomas. In cervical cancer, useful antigens could be encoded byhuman papilloma virus. Chromagranin-A and synaptophysin are antigens ofneuroendocrine and neuroectodermal tumors. Of particular interest areaggressive tumors that form solid tumor masses having necrotic areas.The lysis of such necrotic cells is a rich source of antigens forantigen-presenting cells, and thus the subject compositions and methodsmay find advantageous use in conjunction with conventional chemotherapyand/or radiation therapy.

Tumor-associated antigens can be prepared by methods well known in theart. For example, these antigens can be prepared from cancer cellseither by preparing crude extracts of cancer cells (e.g., as describedin Cohen et al., Cancer Res., 54:1055 (1994)), by partially purifyingthe antigens, by recombinant technology, or by de novo synthesis ofknown antigens. The antigen may also be in the form of a nucleic acidencoding an antigenic peptide in a form suitable for expression in asubject and presentation to the immune system of the immunized subject.Further, the antigen may be a complete antigen, or it may be a fragmentof a complete antigen comprising at least one epitope.

Antigens derived from pathogens known to predispose to certain cancersmay also be advantageously utilized in conjunction with the compositionsand methods provided herein. It is estimated that close to 16% of theworldwide incidence of cancer can be attributed to infectious pathogens,and a number of common malignancies are characterized by the expressionof specific viral gene products. Thus, the inclusion of one or moreantigens from pathogens implicated in causing cancer may help broadenthe host immune response and enhance the prophylactic or therapeuticeffect of the cancer vaccine. Pathogens of particular interest for useherein include the hepatitis B virus (hepatocellular carcinoma),hepatitis C virus (heptomas), Epstein Barr virus (EBV) (Burkittlymphoma, nasopharynx cancer, PTLD in immunosuppressed individuals),HTLV1 (adult T cell leukemia), oncogenic human papilloma viruses types16, 18, 33, 45 (adult cervical cancer), and the bacterium Helicobacterpylori (B cell gastric lymphoma).

Also contemplated herein are pathogen antigens derived from infectiousmicrobes such as virus, bacteria, parasites and fungi and fragmentsthereof, in order to increase lymphocyte activity in response to activeinfection or improve the efficacy of prophylactic vaccine therapy.Examples of infectious virus include, but are not limited to:Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1 (alsoreferred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and otherisolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitisA virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g. strains that cause gastroenteritis);Togaviridae (e.g. equine encephalitis viruses, rubella viruses);Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow feverviruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g.vesicular stomatitis viruses, rabies viruses); Coronaviridae (e.g.coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g.parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis Bvirus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae herpessimplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus(CMV), herpes virus; Poxyiridae (variola viruses, vaccinia viruses, poxviruses); and Iridoviridae (e.g. African swine fever virus); andunclassified viruses (e.g. the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e. Hepatitis C); Norwalk and related viruses, andastroviruses).

Also, gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to Pasteurella species, Staphylococci species, and Streptococcusspecies. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species, and Salmonella species. Specificexamples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfuenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Examples of pathogens also include, but are not limited to, infectiousfungi that infect mammals, and more particularly humans. Examples ofinfectious fingi include, but are not limited to: Cryptococcusneoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomycesdermatitidis, Chlamydia trachomatis, Candida albicans. Examples ofinfectious parasites include Plasmodium such as Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax. Otherinfectious organisms (i.e. protists) include Toxoplasma gondii.

Other medically relevant microorganisms that serve as antigens inmammals and more particularly humans are described extensively in theliterature, e.g., see C. G. A Thomas, Medical Microbiology, BailliereTindall, Great Britain 1983, the entire contents of which is herebyincorporated by reference. In addition to the treatment of infectioushuman diseases, the compositions and methods of the present inventionare useful for treating infections of nonhuman mammals. Many vaccinesfor the treatment of non-human mammals are disclosed in Bennett, K.Compendium of Veterinary Products, 3rd ed. North American Compendiums,Inc., 1995.

Treatment of Cancer

The present invention also provides immunotherapeutic methods fortreating cancer, comprising administering to a patient a therapeuticallyeffective amount of a BTLA-HVEM antagonist, either alone or incombination with other therapeutic compositions.

In a preferred embodiment, immunization is done to promote atumor-specific T cell immune response. In this embodiment, a BTLA-HVEMantagonist is administered in combination with a tumor-associatedantigen. The combination of a tumor-associated antigen and a BTLA-HVEMantagonist promotes a tumor specific T cell response, in which T cellsencounter reduced negative costimulatory signals mediated by BTLA ascompared to those in the absence of the BTLA-HVEM antagonist.

In one aspect, the present invention provides a medicament for thetreatment of cancer, wherein the medicament comprises a BTLA-HVEMantagonist. Also provided are methods for making a medicament useful forthe treatment of cancer, which medicament comprises a BTLA-HVEMantagonist.

Treatment of Autoimmune Disease, Allergy, and Asthma

The present invention also provides methods for inhibiting autoimmuneresponses and treating autoimmune diseases, comprising administering toa patient a therapeutically effective amount of a BTLA-HVEM agonist.Without being bound by theory, administration of a therapeuticallyeffective amount of a BTLA-HVEM agonist inhibits the activity ofautoreactive T and B cells that specifically recognize autoantigens andotherwise negatively affect the physiology of cells that bear them.

Autoimmune disease as used herein includes Rheumatoid arthritis, type 1diabetes, autoimmune thyroiditis, and Lupus. Additional autoimmunediseases are described, for example, in Mackay et al., NEJM,345:340-350, 2001.

In one aspect, the present invention provides a medicament for thetreatment of autoimmune disease, wherein the medicament comprises aBTLA-HVEM agonist. Also provided are methods for making a medicamentuseful for the treatment of autoimmune disease, which medicamentcomprises a BTLA-HVEM agonist.

In another aspect, the invention provides methods for preventing orreducing an allergic reaction in a patient, comprising administering toa patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the present invention provides a medicament for thetreatment or prevention of allergy, wherein the medicament comprises aBTLA-HVEM agonist. Also provided are methods for making a medicamentuseful for the treatment or prevention of allergy, which medicamentcomprises a BTLA-HVEM agonist.

In one aspect, the invention provides methods for reducing the severityof an asthmatic reaction in a patient, comprising administering to apatient a therapeutically effective amount of a BTLA-HVEM antagonist.

In one aspect, the invention provides methods for shortening theduration of an asthmatic reaction in a patient, comprising administeringto a patient a therapeutically effective amount of a BTLA-HVEMantagonist.

In one aspect, the invention provides methods for improving recoveryfrom an asthmatic reaction in a patient, comprising administering to apatient a therapeutically effective amount of a BTLA-HVEM antagonist.

Increasing Graft Survival

The present invention also provides methods for inhibiting a host immuneresponse to transplanted tissue, comprising administering to a patientreceiving transplanted tissue a therapeutically effective amount of aBTLA-HVEM agonist. Administration of the therapeutically effectiveamount of the BTLA-HVEM agonist inhibits the host immune response to thetissue and prolongs its survival.

In one aspect, the present invention provides a medicament for use as animmunosuppressant, wherein the medicament comprises a BTLA-HVEM agonist.Also provided are methods for making such a medicament.

All references cited herein are expressly incorporated herein in theirentirety by reference.

EXPERIMENTAL BTLA Antibodies

Armenian hamsters and Balb/c BTLA KO mice were immunized withoxidatively refolded BI/6 BTLA tetramer protein. The ability of antibodyto block binding of BTLA tetramer to HVEM was determined.

Allelic Clone Isotype Specificity Blocking? Applications 6A6 Hamster IgGBI/6 only Yes FACS, IP 6F7 Mouse IgG1, Balb/c and BI/6 Yes FACS, IP, WBkappa 6G3 Mouse IgG1, Balb/c and BI/6 Yes FACS, IP kappa 6H6 Mouse IgG1,Balb/c and BI/6 Weak FACS, IP kappa 8F4 Mouse IgG1, Balb/c and BI/6 YesFACS, IP kappa 3F9.D12 Mouse IgG1, Balb/c and BI/6 Yes FACS, IP kappa3F9.C6 Mouse IgG1, BI/6 only No FACS, IP, WB kappa

Yeast Display Data: The ability of antibody to bind to BTLA mutants wasdetermined. + indicates binding.

BI/6 BTLA Mutations 6A6 3F9.C6 6G3 6F7 3F9.D12 P41E + + + + +T45N + + + + + P41E, T47K + + + + + P41E, Q52H + + + + + P41E, R55W +− + + + P41E, Q63E − + + + + P41E, C85W +/− + +/− +/− +/− P41E,S91G + + + + + P41E, Q102R − + + + + P41E, T143R + + + + + WEHI.2 − − −− −(I) BTLA Ligand Binding and BTLA Activation

B7 molecules bind to MYPPPY motif on CD28 and CTLA4 within FG loop. Forexample, PD-L1 and PD-L2 bind to FG loop of PD-1. The FG loop of BTLA isfour amino acids shorter than FG loop in CD28/CTLA4, and epitope mappingplaces HVEM interaction towards ‘MBA’ face of Ig fold.

BTLA Binds Naive T Cells but does not Bind B7x

To test for direct interactions between BTLA and B7x, we made NIH 3T3cell lines stably expressing the extracellular domains of B7x, BTLA, andprogrammed death 1 (PD-1) and its ligand (PD-L1), and stained the cellswith PD-1 and BTLA tetramers and with PD-L1 and B7x Fc fusion proteins.Whereas PD-1 tetramer bound to cells expressing PD-L1, as expected, theBTLA tetramer did not bind to cells expressing PD-L1 or B7x.Furthermore, our B7x-Fc fusion protein did not bind cells expressingBTLA.

To identify potential ligands on normal lymphocytes, we next used aBTLA-Fc fusion protein and BTLA tetramer to stain splenocytes (FIGS. 1and 2). As a control, the PD-L1-Fc fusion protein showed selectivebinding to activated but not resting CD4+ T cells and B220+ B cells(FIG. 1), as expected and consistent with the reported inducibility ofPD-1 expression. Notably, our BTLA-Fc fusion protein showed specificbinding to resting CD4+ and CD8+ T cells but not to B220+ B cells (FIG.1). In addition, this binding was greatly reduced after T cellactivation by treatment with antibody to CD3 (anti-CD3) but was notaffected by B cell activation. As B7x is not reported to be expressed bynaive T cells, the binding of the BTLA-Fc fusion protein to T cells isnot consistent with an interaction with B7x. However, we independentlyconfirmed that B7x was not expressed by T cells by examining theexpression of B7x and several other CD28-B7 family members. B7x mRNA wasmost highly expressed in heart and lung but was absent from spleen,lymph node and naive CD4+ T cells. In contrast, we confirmed theexpected lymphoid-specific expression pattern for several CD28-B7 familymembers, including ICOS, PD-1 and BTLA.

To confirm and characterize the potential ligand on T cells identifiedby the BTLA-Fc fusion protein, we next analyzed the properties of BTLAtetramers binding to lymphocytes (FIG. 2). The BTLA tetramer showedstrong binding to both CD4+ and CD8+ T cells obtained from both spleenand lymph nodes and bound weakly to non-T lymphocytes (FIG. 2 a).Furthermore, binding to T cells was reduced after anti-CD3 stimulation(FIG. 2 b), similar to our results obtained with BTLA-Fc fusion proteins(FIG. 1). In contrast, treatment of splenocytes with antiimmunoglobulinM (anti-IgM) or lipopolysaccharide did not reduce BTLA tetramer stainingof CD4+ T cells. BTLA tetramers showed very slight binding to restingand activated B cells (FIG. 2 b). We also examined BTLA tetramerstaining in thymic subsets (FIG. 2 c). BTLA tetramer staining was lowestin CD4+ CD8+ (double-positive) thymocytes and showed more staining inmature CD4+ or CD8+ thymocytes and double-negative (CD4−CD8−)thymocytes, again indicating some physiological regulation of the BTLAligand. As BTLA tetramer binding was modulated 48 h after anti-CD3stimulation of T cells, we did a more detailed kinetic analysis usingDO11.10 T cells activated in vitro with ovalbumin (OVA) peptide (FIG.3). Again, BTLA tetramer binding was regulated during activation,initially increasing by twofold at 24 and 48 h after antigen-specificstimulation, decreasing on day 3 and day 4, and increasing again by day7. Expression of this BTLA ligand was similar in both T helper type 1-and T helper type 2-inducing conditions. Thus, BTLA tetramers andBTLA-Fc fusion proteins have very similar binding properties tolymphocytes, and a BTLA ligand is expressed by resting T cells andundergoes regulation during thymocyte development and T cell activation.

Cloning a BTLA-Interacting Protein

We constructed a retroviral cDNA library from lymphocytes and transducedtwo host cell lines, BJAB and NIH 3T3, that were negative for BTLAtetramer binding (FIG. 4 a). After four successive rounds of sorting, weobtained lines uniformly positive for BTLA tetramer staining, which weused to amplify retrovirus-specific inserts. From BJAB cells, weobtained a predominant RT-PCR product that we identified as mouse HVEM.From NIH 3T3 cells we also obtained mouse HVEM as the main component ofRT-PCR isolates. Among the minor retroviral inserts identified from NIH3T3 cells, 4-1BB was the only transmembrane receptor; it also belongs tothe TNFR superfamily.

We next tested these isolates as candidates for direct interactions withBTLA tetramers. We expressed full-length cDNA clones of mouse HVEM,human HVEM, mouse 4-1 BB and mouse LTβR, which binds the same ligands(LIGHT and LTa) as HVEM, in BJAB cells and analyzed these cells forbinding to BTLA tetramers. We specifically constructed BTLA tetramersfrom both the C57BL/6 and BALB/c alleles to identify any potentialallelic differences in binding (FIG. 4 b). We found specific binding ofboth forms of BTLA tetramers to green fluorescent protein (GFP)-positiveBJAB cells expressing mouse HVEM but not to BJAB cells expressing humanHVEM, mouse 4-1 BB or mouse LTβR or to GFP-negative uninfected BJABcells.

HVEM Induces BTLA Phosphorylation

We next sought to determine if HVEM could induce BTLA phosphorylation(FIG. 4 c, d). We analyzed BTLA phosphorylation in EL4 cells usingimmunoprecipitation immunoblot analysis as described above. EL4 cellshad low expression of BTLA but no detectable HVEM, as assessed by BTLAtetramer binding. We therefore examined EL4 cells for BTLAphosphorylation and SHP-2 coimmunoprecipitation after contact with mouseHVEM expressed by BJAB cells. EL4 cells alone showed neithercoimmunoprecipitation of SHP-2 with BTLA (FIG. 4 c) nor direct tyrosinephosphorylation of BTLA (FIG. 4 d). Mixing of EL4 cells withHVEM-expressing BJAB cells induced both coimmunoprecipitation of SHP-2with BTLA and tyrosine phosphorylation of BTLA. In contrast, mixing EL4cells with HVEM-negative BJAB cells induced neithercoimmunoprecipitation of SHP-2 with BTLA nor BTLA phosphorylation. Ascontrols, pervanadate treatment of EL-4 cells inducedcoimmunoprecipitation of SHP-2 and tyrosine phosphorylation of BTLA, butBJAB cells alone, either. HVEM-negative or expressing HVEM, showedneither SHP-2 coimmunoprecipitation nor BTLA phosphorylation. Thus,these results show that HVEM can induce BTLA tyrosine phosphorylationand association with SHP-2.

HVEM-BTLA Interactions are Conserved in Human

Because tetramers of mouse BTLA bound mouse HVEM but not human HVEM, wesought to determine if the BTLA-HVEM interaction was conserved inhumans. Therefore, we generated a human BTLA-Fc fusion protein andcharacterized its interactions with mouse and human HVEM (FIG. 4 e). Themouse BTLA-Fc fusion protein bound to BJAB cells expressing mouse HVEMbut not cells expressing human HVEM, confirming the data obtained withmouse BTLA tetramers (FIG. 4 b). In addition, the human BTLA-Fc fusionprotein bound to BJAB cells expressing human HVEM (FIG. 4 e). The humanBTLA-Fc fusion protein also bound, although more weakly, to BJAB cellsexpressing mouse HVEM. These interactions were specific, as the isotypecontrol antibody and the B7x-Fc fusion protein did not bind to BJABcells expressing either mouse or human HVEM. Thus, the interactionbetween BTLA and HVEM occurs in human lymphocytes, as it does in mouselymphocytes. Also, although cross-species interactions are noted forhuman BTLA and mouse HVEM (FIG. 4 e), it seems that this cross-speciesinteraction is weaker than the intraspecies interaction.

BTLA Interacts with the CRD1 Region of HVEM

HVEM is a member of the TNFR superfamily and interacts with the twoknown TNF family members LIGHT and LTα. Because HVEM has multipleligands, we sought to determine whether we could detect additionalligands for BTLA. Thus, we compared binding of BTLA tetramers towild-type and HVEM-deficient lymphocytes (FIG. 5 a). BTLA tetramersshowed no detectable specific binding to HVEM-deficient CD4+ or CD8+ Tcells but showed the expected binding to wild-type T cells. Even the lowbinding of BTLA tetramer to B cells was reduced to undetectable amountsin HVEM-deficient B cells (FIG. 5 a). Thus, we found no evidence ofadditional ligands for BTLA in mice.

The interaction between HVEM and LIGHT can be detected with an HVEM-Fcfusion protein containing the four extracellular CRD regions of HVEMfused to the Fc region of human IgG1. We therefore sought to determinewhether this HVEM-Fc fusion protein can also bind BTLA (FIG. 5 b).Because LIGHT is expressed by CD11c+DCs but not by B220+ B cells, wecompared the binding of HVEM-Fc fusion protein to B cells and DCs fromwild-type and BTLA-deficient mice (FIG. 5 b). The HVEM-Fc fusion proteinbound to wild-type B cells but not to BtIa−/− B cells. In contrast, theHVEM-Fc fusion protein bound to wild-type DCs with only slightly reducedbinding to BtIa−/− DCs. We next compared the binding of HVEM-Fc fusionprotein to wild-type and LIGHT-deficient (Tnfsf14−/−) B cells and DCs(FIG. 5 c). The HVEM-Fc fusion protein bound to wild-type and TnfsfU−/−B cells and DCs with nearly equal intensity. In addition, HVEMexpression was actually increased in BtIa−/− mice compared with that inwild-type mice. This result might indicate that endogenous HVEMexpression is regulated by interaction with BTLA, similar to thereported regulation of HVEM expression by LIGHT. Furthermore, thisresult formally shows that HVEM expression does not require BTLA as a‘chaperone’. These results might suggest that BTLA is the only ligandfor HVEM on B cells, but such conclusions based solely on solublestaining reagents may be misleading, and it is possible that HVEM couldalso interact with other unknown molecules on B cells. For DCs, it seemsthat both BTLA and LIGHT are ligands for HVEM.

We sought to identify which domains of HVEM are involved in BTLAinteractions. HVEM has four extracellular CRDs; it binds LIGHT and LTathrough CRD2 and CRD3 and binds herpes glycoprotein D through CRD1. Weconstructed a series of HVEM mutants, including a mouse HVEM GFP fusionprotein, an HVEM deletion mutant lacking the N-terminal CRD1 as a GFPfusion protein, an intact human HVEM, and a chimeric HVEM containingmouse CRD1 linked to human CRD2. We expressed this panel of HVEM mutantsin BJAB cells and examined binding of the mouse BTLA tetramer (FIG. 5d). As expected, the BTLA tetramer did not bind uninfected BJAB cellsbut bound to wild-type mouse HVEM. However, the mouse BTLA tetramer didnot bind to the HVEM mutant lacking CRD1. In addition, BTLA tetramer didnot bind to human HVEM but did bind to the mouse-human chimeric HVEM(FIG. 5 d). As a control, we assessed the amounts of human HVEMexpressed by these cell lines (FIG. 5 d), confirming expression of thehuman and chimeric HVEM molecules. These results indicate an importantfunction for the CRD1 domain of mouse HVEM for BLTA interactions but donot exclude the possibility of a contribution by other domains.

HVEM Inhibits Antigen-Driven T Cell Proliferation

HVEM is expressed by several types of cells, including T cells, B cellsand DCs, complicating the analysis of potential interactions betweencells expressing LIGHT, BTLA and HVEM. Thus, we first sought to confirmthe reported costimulatory effects of LIGHT on CD4+ T cells in oursystem. We stimulated highly purified CD4+ T cells with increasingamounts of anti-CD3 in the presence of various concentrations ofplate-bound LIGHT (FIG. 6 a). At suboptimal concentrations of anti-CD3stimulation, LIGHT strongly augmented T cell proliferation in adose-dependent way. At the highest dose of anti-CD3, the costimulatoryeffect of LIGHT was reduced slightly because of an increase in theLIGHT-independent proliferation. These data confirm reports that LIGHTengagement of HVEM provides positive costimulation.

We next tested whether BTLA or HVEM expression by antigen-presentingcells (APCs) inhibited or activated T cells. For this, we produced apanel of Chinese hamster ovary (CHO) cells expressing variouscombinations of 1-Ad and B7-1 plus either BTLA or HVEM using retrovirustransduction and cell sorting. We confirmed expression of 1-Ad, B7-1,BTLA and HVEM by these cell lines using flow cytometry. We sought todetermine if BTLA expression by APCs costimulated DO11.10 T cells (FIG.6 b). CHO cells expressing I-Ad alone supported minimal T cellproliferation, similar to that seen with T cells and peptide alone. As apositive control, CHO cells expressing I-Ad and B7-1 supported higherproliferation in response to OVA peptide. In contrast, BTLA expressionby APCs did not augment T cell proliferation induced by CHO cellsexpressing I-Ad alone (FIG. 6 b), as did expression of B7-1, suggestingthat BTLA does not provide costimulation to T cells through HVEMengagement.

Whereas BTLA, unlike LIGHT, may not activate HVEM, HVEM seems toactivate BTLA, as evidenced by BTLA phosphorylation and SHP-2association (FIG. 4 c, d). Thus, we sought to determine whether HVEMexpression by APCs influenced T cell proliferation (FIG. 6 c). Thepeptide dose-dependent proliferation supported by CHO cells expressingI-Ad alone was reduced when HVEM was coexpressed on these CHO cells(FIG. 6 c). Furthermore, as expected, B7-1 increased T cellproliferation induced by peptide and I-Ad (FIG. 6 d), shifting thedose-response to lower concentrations of peptide. Again, coexpression ofHVEM on these CHO cells reduced peptide-dependent T cell proliferation.The inhibition produced by HVEM at the highest peptide concentrationswas smaller than the inhibition seen with intermediate stimulation.

We extended this analysis using T cells labeled with carboxyfluoresceindiacetate succinimidyl diester (CFSE; FIG. 7). In addition, we testedwhether the inhibitory effect of HVEM on T cell proliferation requiredBTLA by using BtIa−/− DO11.10 T cells. Using CHO cells lacking B7-1expression, we did not note T cell proliferation at the lowest dose ofOVA peptide (0.03 μM) on days 3 and 4 (FIG. 7 a). However, higherpeptide concentrations (0.3 μM) induced T cell proliferation on days 3and 4. In these conditions, expression of BTLA on CHO cells had noeffect on T cell proliferation at anytime. However, expression of HVEMon CHO cells greatly reduced T cell proliferation, which occurred onlyin wild-type DO11.10 T cells, not BtIa−/− T cells, and was evident ondays 3 and 4 after activation.

We next examined the effects of HVEM on T cell proliferation in responseto antigen presentation by CHO cells expressing B7-1 (FIG. 7 b). Again,B7-1 increased T cell proliferation induced by peptide and I-Ad,shifting the dose-response to lower concentrations of peptide, asdemonstrated by larger numbers of cellular divisions at lower doses ofpeptide; this was clearly evident on day 3 as well as day 4. In theseconditions, coexpression of BTLA on CHO cells had no effect on T cellproliferation. In contrast, coexpression of HVEM on CHO cells caused areduction in proliferation of wild-type DO11.10 T cells, but this wasevident only at the lowest peptide dose and was evident only on day 3,not day 4, after T cell activation. This inhibition of T cellproliferation was specific to BTLA, as we found it only in wild-type butnot BtIa−/− T cells. In summary, HVEM inhibits bothcostimulation-independent and costimulation-dependent proliferation, butis more effective in blocking activation of antigen stimulated T cellsat low B7-1 expression. Furthermore, HVEM-mediated inhibition of T cellproliferation requires BTLA expression by T cells.

Methods (I)

Mice

C57BL/6 and BALB/c mice (Jackson Labs) were bred in our facility.BtIa−/− mice were backcrossed to BALB/c for nine generations and weresubsequently crossed onto the DO11.10 T cell receptor-transgenicbackground. LIGHT-deficient mice were previously described andHVEM-deficient (Tnfrsf14−/−) mice will be described elsewhere.

Plasmids and Retroviral Constructs.

The sequences of all oligonucleotides are provided in Sedy et al.,Nature Immunology (2005) 6:90-98. For preparation of B7x-B7h-GFP-RV, aPCR product made with primers 5′Bgl2 mB7x and B7xB7h bottom using IMAGEcDNA clone 3709434 as the template, plus a PCR product made with primersB7xB7h top and 3′R1GFP using the B7h-GFP plasmid (a gift from W. Sha,University of California, Berkeley, Calif.) as the template, wereannealed and amplified with Pfu polymerase with primers 5′Bgl2 mB7x and3′R1GFP. This product, encoding the B7x extracellular domain, B7htransmembrane and cytoplasmic domains fused to GFP, was digested withBglII and EcoRI and was cloned into IRES− GFP-RV that had been digestedwith BglII and EcoRI.

The plasmid huHVEM-IRES-GFP-RV was produced by amplification of huHVEMwith primers 5′Bgl2 huHVEM and 3′Xho1 huHVEM using IMAGE cDNA clone5798167 (Invitrogen) as the template, followed by digestion with BglIIand XhoI and ligation into Tb-lym-IRES-GFP-RV that had been digestedwith BglII and XhoI, replacing the Tb-lym cDNA with that of huHVEM.Similarly, m4-1BB-IRES-GFP-RV was prepared with primers 5′Bgl2 m4-1BBand 3′Xho1 m4-1BB using library plasmid as the template, followed bydigestion with BglII and XhoI and ligation into Tb-lym-IRES-GFP-RV. Theplasmid mLT R-IRES-GFP-RV was prepared with primers 5′Bgl2 mLT R and3′Sal1 mLT R using IMAGE cDNA clone 5293090 (Invitrogen) as thetemplate, followed by digestion with BglII and SalI and ligation intoTb-Iym-IRES-GFP-RV. The plasmid mHVEM-FL-IRES-GFP-RV was similarlyprepared with primers 5′Bgl2 mHVEM and 3′Xho1 mHVEM using, as thetemplate, cDNA from library infected BJAB cells sorted for BTLA tetramerbinding, followed by digestion with BglII and XhoI and ligation intoTb-lym-IRES-GFP-RV. Three amino acid changes (N58S, K92R and E128G) inmouse HVEM cDNA cloned from the retrovirus library, compared with thatof mouse HVEM cDNA from the 129 SvEv mouse strain, were implemented byQuick Change mutagenesis (Stratagene) to generate mHVEM(129)-IRES-GFP-RVwith serial application of the primers S—N top plus S—N bot; R-K topplus R-K bot; and G-E top plus G-E bot.

The plasmid mHVEM-FL-GFP-RV was made from two PCR products, with primers5′Bgl2 mHVEM and mHVEM/GFP bot using mHVEM-FL-IRES-GFP-RV as thetemplate, and primers mHVEM/GFP top and 3′GFP+Sal usingmHVEM-FL-IRES-GFP-RV as the template; the PCR products were annealed,amplified with primers 5′Bgl2 mHVEM and 3′GFP+Sal, digested with BglIIand SalI and ligated into IRES-GFP-RV that had been digested with BglIIand SalI. The plasmid mHVEM-FL-GFP-RVCRD1 was made by Quick Changemutagenesis from mHVEM-FL-GFP-RV with primers mHVEM d1 top and mHVEM d1bot. The plasmid m/hHVEM-IRES-GFP-RV (mouse CRD1 fused to human CRD2)was made from two PCR products, with primers 5′Bgl2 mHVEM and m/hHVEMbot using mHVEM-FL-IRES-GFP-RV as the template, and primers m/hHVEM topand 3′Xho hHVEM using hHVEM-IRES-GFP-RV as the template; the PCRproducts were annealed, amplified with primers 5′Bgl2 mHVEM and 3′XhohuHVEM, digested with BglII and XhoI and ligated into Tb-lym-IRES-GFP-RVthat had been digested with BglII and XhoI. C57BL/6-BTLA-GFP-RV, aBTLA-GFP chimera, was prepared from two PCR products, with primersJ10RV1 (Bgl 2) and 3′J10+10 using C57BL/6 BTLA cDNA as the template, andprimers 5′GFP+10 and 3′GFP+Sal using GFP cDNA as the template; the PCRproducts were annealed, amplified with J10RV1 (Bgl 2) and 3′GFP+Sal,digested with Bglll and SalI and ligated into Tb-lym-IRES-GFP-RV thathad been digested with BglII and XhoI. A cytoplasmic deletion of thisconstruct, BTLA-trunc-GFP-RV, was made by site-directed mutagenesis(Stratagene) with primers mj11trunc top and mj11trunc bottom.

PD-1-GFP-RV was made by amplification of the PD-1 coding region withprimers PD15′ and PD13′ using PD-1 cDNA as the template (a gift from T.Honjo, Kyoto University, Kyoto, Japan); the PCR product was digestedwith BglII and BamHI and was cloned into AIB3-GFP MSCV that had beendigested with BglII and BamHI (a gift from W. Sha). Similarly,PD-L1-GFP-RV was made by amplification of the region encoding PD-L1 withprimers PD-L1G5′ and PD-L1G3′ using PD-L1 cDNA (a gift from T. Honjo) asthe template; the PCR product was digested with BglII and BamHI and wasligated into AIB3-GFP MSCV.

PD-1 pET28 was made by amplification of the immunoglobulin domain ofPD-1 with primers PD1Tet5′ and PD1Tet3′ using PD-1-GFP-RV plasmid as thetemplate, followed by digestion with NcoI and BamHI and ligation intoMLL1-pET28 (a gift from D. Fremont, Washington University, St. Louis,Mo.) that had been digested with NcoI and BamHI. Similarly, B6-BTLApET28 was made by amplification of the extracellular immunoglobulindomain of BTLA with primers J11TetMus5′ and J11TetB63′ using C57BL/6BTLA-GFP-RV plasmid as the template, followed by digestion with NcoI andBamHI and ligation into MLL1-pET28. Similarly, BALB-BTLA pET28 was madewith primers J11TetMus5′ and J11TetWEHI3′ using mJ11W1 as the template,and digestion with NcoI and BamHI and ligation into MML1-pET28. Theimmunoglobulin domain was ‘corrected’ to authentic BALB/c allelicsequence (data not shown) by serial mutagenesis with primers W1e23k5′and W1e23k3′ followed by primers W1h38n3B and W1h38n5C.

Fc Fusion Proteins

For the creation of CD47-Fc-αTP-ires-GFP-RV, a bicistronic retroviralvector for Fc fusion proteins, CP318 (a gift from Lewis Lanier,University of California, San Francisco, Calif.) was digested with PfIFI and NotI, treated with Vent polymerase and ligated intom1L-12R-ires-GFP-RV that had been digested with BglII and XhoI andtreated with mung bean nuclease. The plasmids mBTLA-Fc-αTP-ires-GFP-RV,mB7x-Fc-αTP-ires-GFP-RV, mPD-L1-Fc-αTP-ires-GFP-RV andhBTLA-Fc-ocTP-ires-GFP-RV were made by ligation of the followingXhoI-digested PCR products containing the immunoglobulin domains regionsof these genes into the XhoI site of CD47-Fc-αTP-ires-GFP-RV. Theproduct mBTLA was made with primers 5′xho mJl 1 dodecamer and 3′xho mJ11 dodecamer using as a template the C57BL/6 splenocyte phage library(Stratagene). The product mB7x was made with primers 5′xho mB7xdodecamer and 3′xho mB7x dodecamer using IMAGE cDNA clone 3709434(Invitrogen) as the template. PD-L1 was made with primers 5′xho mPDL2dodecamer and 3′Xho PDLL dodecamer using pBacPAK8-PDL1 (a gift from T.Honjo) as the template. Human BTLA was made with primers 5′Xho hJ11 Igand 3′Xho hJH Ig using hJ11(corr)ires-GFP-RV as the template.

Fc fusion proteins were produced by transfection of Phoenix E cells,were purified with Affi-prep protein A columns (Biorad) and weredialyzed against PBS and stored at −70° C. For flow cytometry, cellswere stained with 200 ng of purified Fc-fusion protein or, for hBTLA-Fcfusion protein, 1 ml of supernatant, followed byphycoerythrin-conjugated anti-human IgG (heavy plus light) that had beenadsorbed against proteins from mouse, rat, cow and other species(Jackson Immunoresearch), and anti-mCD4-tricolor (Caltag) andanti-mB220-fluorescein isothiocyanate (FITC; BD-Pharmingen).

Production of Tetramers

Tetramers produced with plasmid PD-1 pET28, B6-BTLA pET28 or BALB-BTLApET28 were transformed into BL21-CodonPlus (DE3) RIPL Competent Cells(Stratagene). Purified proteins were biotinylated in vitro with BirAligase (Avidity), purified by gel filtration and concentrated. Tetramerswere formed by the addition of biotinylated protein tostreptavidin-phycoerythrin at a molar ratio of 1:4.

Cell Lines

BJAB and NIH 3T3 cells were from A. Chan (Washington University, St.Louis, Mo.); EL-4 cells were from T. Ley (Washington University, St.Louis, Mo.); 293T cells were from R. Schreiber (Washington University,St. Louis, Mo.); CHO cells were from A. Sharpe (Harvard University,Boston, Mass.); and Phoenix A and E packaging cells were from AmericanType Culture Collection. Retrovirus constructs were packaged either inPhoenix A or E cells by calcium phosphate transfection. CHO cells weretransduced by retrovirus packaged by transfection of 293T cells withpYITG plus pCGP (a gift from W. Sha) and were sorted for GFP to morethan 95% purity, followed by staining with 6A6 (anti-BTLA) orBTLA-phycoerythrin tetramers.

Retrovirus Library

Purified BALB/c and C57BL/6 splenocytes were left unstimulated or wereactivated for 48 h with plate-bound anti-CD3 (500A.2 ascites) or solubleanti-IgM (Jackson Immunoresearch), then RNA was purified (RNeasy minikit; Qiagen) and mRNA was made with the Nucleotrap mRNA purification kit(Clontech), full-length cDNA was made with the SMART cDNA LibraryConstruction Kit (Clontech) and double-stranded cDNA was made bylong-distance PCR with 5′PCR primer and CDS III/3′PCR primer; the PCRproducts were digested with SfiI, size fractionated, amplified cDNAligated into Sfi1-digested MSCV-ires-Thy1.1 retrovirus vector (a giftfrom W. Sha) and were transduced into XL-10 gold (Stratagene) for alibrary transcript complexity of 2×10⁶. The library plasmid was purifiedwithout further amplification by CsCl gradient ultracentrifugation.Infected NIH-3T3 cells (8×10⁶) and infected BJAB cells (6×10⁶) weregenerated from retrovirus made by calcium phosphate transfection ofPhoenix E cells; the total number of infected cells was assessed byanti-Thy1.1-FITC (eBioscience) staining. Serial rounds of cell sortingused anti-Thy1.1-FITC and BALB/c and C57BU6 BTLA tetramers. When thesorted cells were more than 80% positive for Thy1.1 and BTLA tetramer,RNA was prepared and reverse-transcribed, cDNA was amplified with Taqpolymerase and primers Sfi 5′ and Sfi 3′, and PCR products were clonedinto pGEM-T Easy (Promega).

T Cell Purification and Stimulation

T cells were purified (>90%) with anti-CD4 magnetic beads (Miltenyi)and, where indicated (FIGS. 6 b-d, 7) by subsequent sorting forpopulations that were negative for B220-FITC and CD11c-phycoerythrin andpositive for CD4-CyChrome (>98%). For T cell stimulation with anti-CD3and LIGHT, 2C11 (BD Pharmingen) was coated onto 96-well plates, followedby LIGHT (PeproTech) at the indicated doses (FIG. 6 a). Purified T cellswere plated at a density of 1×10⁶ cells/ml in 100 μA media per well. CHOcells were treated in media for 16 h at 37° C. with 50 μg/ml ofmitomycin C (Sigma), were washed twice in PBS and were plated at adensity of 1×10⁶ cells/ml in 100 μA media in 96-well plates forproliferation assays or in 1 ml media in 24-well plates for CFSEanalysis. For proliferation assays, purified T cells were plateddirectly onto CHO cells at a density of 1×10⁶ cells/ml in 100 μl mediaand OVA peptide. After 48 h, cells were pulsed for 12 h with 1 μCi/wellof [³H]thymidine. For CFSE analysis, purified T cells were washed threetimes with PBS, were incubated for 8 min at 20° C. with 104 CFSE(Molecular Probes), were ‘quenched’ with fetal calf serum, were washedtwice with media and were plated directly onto CHO cells at a density of1×10⁶ cells/ml in 1 ml media plus OVA peptide. After 3 and 4 d, cellswere stained with CD4-FITC and were analyzed by flow cytometry.

Immunoblot Analysis

For cell-mixing experiments, 25×10⁶ EL4 cells were mixed with 25×10⁶BJAB cells expressing GFP or 25×10⁶ BJAB cells expressing mouse HVEM in1 ml for 4 min at 37° C. and were lysed. Extracts were precleared withprotein G-Sepharose (Pharmacia), followed by immunoprecipitation with 9μg of 6A6 (anti-mBTLA) or isotype control Armenian hamster IgG (SantaCruz) and 40 μl protein G-Sepharose (Pharmacia), then were washed andanalyzed by SDS-PAGE. Immunobiot analyses for SHP-2 and phosphotyrosinewere done as described (1, 3) in Watanabe et al, Nat. Immunol. (2003)4:670-679 and Gavrieli et al, Biochem. Biophys. Res. Commun.312:1236-1243 (2003).

For further details regarding Example I, including references, see Sedyet al., Nat. Immunol., 6:90-98, which is expressly incorporated hereinin its entirety by reference.

(II) BTLA Polymorphism and BTLA Binding Antibodies

Allelic Polymorphisms in BTLA

We previously generated BTLA cDNA from several sources, including fromthe cell line WEHI 231, a commercial murine C57BL/6 splenocyte cDNAlibrary, and 129SvEv mice, finding several polymorphisms within the BTLAIg domain coding sequence. To determine the basis of differences, wesequenced the coding region for the BTLA Ig domain from genomic DNA ofseveral inbred and wild mouse strains (FIG. 8). Among 23 strains, weidentified three distinct alleles of BTLA, differing in their predictedamino acid sequence and potential predicted disulphide bonding pattern(FIG. 8A). The allele represented by BALB/c was present in CBA/J, SJL/J,New Zealand White (NZW), BXSB, C3H/J, New Zealand Black (NZB/BinJ), NOD,129SvEv, and 129SWJ (FIG. 8B). A second allele, represented by thestrains MLR/lpr, AKR, SWR, CALB/RK, and DBA/2J, differed from the BALB/callele at only one amino acid, containing histidine rather thanasparagine at residue 38 of the BTLA protein. These two alleles eachhave five cysteine residues within the Ig domain, predicting twodisulfide bonds and one unpaired cysteine. The third allele, representedby C57BL/6, was also present in B10.PL and several wild-derived inbredstrains, and differed from the BALB/c and MLR/lpr alleles at 10 and 11amino acid residues, respectively (FIG. 8A). Notably, the C57BL/6 allelehas a cysteine at amino acid residue 49, making six total cysteineresidues with three predicted disulfide bonds in the BTLA Ig domain. Asa control, we found no sequence polymorphisms in the PD-1 Ig domain fromBALB/c, MLR/lpr, and C57BL/6.

Generation of Allele-Specific mAbs to Murine BTLA

To generate anti-BTLA mAbs, we immunized Armenian hamsters and BTLA−/−BALB/c mice with recombinant Ig domain of the C57BL/6 BTLA allele. Toallow the identification of Abs that could potentially recognize eitherthe BALB/c or C57BL/6 allele of BTLA, hybridoma supernatants werescreened for binding to BJAB cells expressing either the C57BL/6 orBALB/c allele of BTLA as a GFP fusion protein. One hamster anti-BTLA Ab,6A6, was identified that reacted only with the C57BL/6, but not theBALB/c, allele of BTLA (FIG. 9A). The majority of the murine anti-BTLAmAbs reacted with both the C57BL/6 and BALB/c BTLA alleles, including6F7, 6G3, 8F4, and 3F9.D12 (FIG. 9B). One murine Ab, 3F9.C6, reactedonly with C57BL/6 BTLA, and not with BALB/c BTLA. Another Ab, 6H6,reacted with both alleles, but stained the C57BL/6 allele more highlythan the BALB/c allele. For each of these Abs, staining was observed onwild type splenocytes, but not splenocytes of BTLA−/− mice (FIG. 9C),suggesting that these Abs in fact recognize BTLA, and react with nativeBTLA as well.

To further assess how these Abs interact with BTLA, we characterizedtheir behavior in IP and Western blot analysis (FIGS. 9D and E). Thepan-specific Abs 6F7 and 6G3 each specifically immunoprecipitated boththe C57BL/6 and BALB/c BTLA-GFP fusions proteins from BJAB cells (FIG.9D, bottom panel). Importantly, the C57BL/6-specific 6A6 Ab didimmunoprecipitate the C57BU6 BTLA allele, but not the BALB/c allele(FIG. 9D, compare lanes 3 and 6), indicating that the allelicspecificity observed by FACS analysis extends to its behavior in IPWestern blot analysis. Also, these interactions seen in IP Western blotanalysis were specific because no BTLA was immunoprecipitated usingmouse or hamster IgG1 as an isotype control (FIG. 9D, lanes 7-10).

Notably, although equivalent amounts of each BTLA allele wereimmunoprecipitated when assessed by immunoblotting for the GFP epitopeof the fusion proteins, detection of the Ig domain by IP Western blotanalysis was not equally efficient. Following immunoprecipitation, theC57BL/6 BTLA Ig domain was detected much more strongly than the BALB/callele by 6G3 and 6F7, both pan-specific anti-BTLA Abs, (FIG. 9D, toppanel, lanes 1, 2, and 4-6). These results may indicate differentialsensitivity between alleles for recognition or detection of the Igdomains, even using pan-specific Abs, which could result fromdifferential sensitivity to denaturation of the antigenic epitope.Whatever the cause, it is necessary to consider this fact when using IPWestern blot analysis in comparing BTLA from varying allelicbackgrounds. Finally, certain Abs allow coimmunoprecipitation ofBTLA-associated proteins. For example, IP Western blot analysis using6A6 reproduces the known specific and inducible coassociation of SHP-2with BTLA following pervanadate treatment (FIG. 9E).

Mapping Antigenic Epitopes Recognized by anti-BTLA Abs

To map which of the polymorphic residues differing between BALB/c andC57BL/6 BTLA were involved in strain-specific reactivity of 6A6 and3F9.C6, we used yeast display technology. We first expressed the BTLA Igdomain as an Aga2 fusion protein, and then generated a series of mutantBTLA Ig domains with single amino acid substitutions at the polymorphicresidues, replacing BALB/c residues into the C57BL/6 allele one residueat a time (FIG. 10). This series of wild type and mutant BTLA proteinswere then analyzed for reactivity with pan-specific anti-BTLA mAbs andtwo B6-specific Abs, 6A6 and 3F9.C6 (FIG. 10). As a positive control, weconfirmed that the pan-specific anti-BTLA mAb 6F7 recognized the wildtype C57BL/6 BTLA Ig domain, and also recognized each of the singleresidue substitutions of BTLA (FIG. 10, left column), as expected forpan-specific reactivity. In contrast, the two C57BL/6-specific Absrecognized some, but not all of BTLA mutants. Specifically, 6A6 showed avery selective loss of reactivity only with the Q27E, C49W, and Q66Rsubstitutions, indicating that these residues are involved in thestrain-specific recognition of BTLA. A distinct pattern of reactivitywas observed with 3F9.C6, with a selective loss of reactivity with theR107W substitution and reduced reactivity with the Q27E substitution.Also, whereas 6A6 reactivity is sensitive to the C49W substitution,which disrupts one of three predicted disulphide bonds, 3F9.C6reactivity remains in this substitution. These results indicate that theC57BL/6 specificity of these two Abs derive from interactions with thedistinct, but polymorphic, region of the BTLA Ig domain.

In summary, at least two of the BTLA alleles can be distinguished bytheir antigenic structure, as shown by two C57BL/6-specific anti-BTLAAbs. Importantly, we also identified several pan-specific anti-BTLA Abs,which now allow direct comparisons of the fine specificity of tissueexpression of native BTLA expression between various murine strains.

Distribution and Expression of Murine BTLA

In our previous studies, we were restricted to analyzing BTLA expressioneither by mRNA expression or by using epitope-tags because we lacked Absto native BTLA. Conceivably, we failed to detect low but physiologicallyimportant levels of BTLA on certain lymphocyte subsets for this reason.Thus, we examined BTLA surface expression on various lymphoid subsetsagain, using both allele-specific Ab 6A6 and pan-specific Ab 6F7 (FIG.11).

First, BTLA was expressed uniformly on B cells at levels that weresimilar for C57BL/6 and BALB/c mice (FIG. 11A). CD4+ and CD8+ T cellsexpressed lower levels of BTLA compared with B cells, but again, atlevels that were similar for C57BL/6 and BALB/c mice. For 6A6, we foundthat a subpopulation of CD11b+ cells, CD11c+ dendritic cells, and DX5+cells were positive for BTLA expression, and again identified only inC57BL/6 cells as expected (FIG. 11A, middle row). Using the pan-specific6F7 Ab, we found that B cells express the highest levels of BTLA, againat levels similar between C57BL/6 and BALB/c mice, with lower levelsexpressed in CD4 and CD8 T cells (FIG. 11A, lower row). Interestingly,using the pan-specific reagent 6F7, we found that BTLA was expressed onCD11c+ BALB/c cells at levels similar to CD11c+ C57BL/6 cells, but thatBTLA was only expressed on CD11b+ macrophages and DX5+ NK cells fromC57BL/6 mice, but not in BALB/c mice (FIG. 11A, lower row). The factthat 6F7 detects BTLA expression on B cells, T cells, and CD11c+ cellsfrom both BALB/c and C57BL/6 mice serves as a control for its ability tobind BTLA from both strains. Thus, the selective binding of 6F7 to DX5+and CD11b+ cells only in C57BL/6, not BALB/c mice, indicates adifference between these strains for BTLA expression by these celltypes. Thus, these strains appear to have a distinct difference in thecell types expressing detectable BTLA, explaining the differencesbetween BTLA expression reported previously (15, 17).

We also examined BTLA expression in splenic B cell populations (FIG.11B). BTLA expression was detected at the highest levels on follicular Bcells (lgMlowCD21/CD35int), and at reduced levels on marginal zone Bcells (lgMhighCD21/CD35high) and transitional B cells(lgMlowCD21/CD35low) (FIG. 11B). Notably, because the 6F7 pan-specificAb was used for analysis, we can also conclude that the levels on eachsubpopulation of B cells are similar between C57BL/6 and BALB/c mice(FIG. 11B).

We next examined BTLA expression in thymocyte and B cell development(FIG. 12). In thymus, BTLA was expressed at highest levels on matureCD4+ T cells, and at slightly reduced levels on CD8+ T cells (FIG. 12A).BTLA expression on immature CD4−CD8− T cells or CD4+ CD8+ doublepositive T cells was nearly undetectable (FIG. 12A). In bone marrow,BTLA was expressed at the highest levels on B220highlgM+ mature B cells(FIG. 12B), and was detected at relatively low levels on B220low/IgM+immature B cells. BTLA expression was undetectable on B220+lgM− pro-Bcells and pre-B cells. Further, we found no differences between C57BL/6or BALB/c mice for the levels of BTLA expression on the thymocyte andbone marrow populations.

Finally, we examined the BTLA expressed on CD4+ T cells under variousconditions of activation and polarization by cytokines (FIG. 13A). BTLAsurface expression on resting CD4+ T cells was induced by 10-fold on day2 following activation with Ag and APCs, decreased by day 4, and wasnearly undetectable by day 7 after activation (FIG. 13A). The rapidincrease in BTLA expression by day 2 on Ag-activated CD4+ T cellsoccurred both in Th1-inducing or Th2-inducing conditions (FIG. 13A).Upon secondary T cell activation, BTLA was again highly induced 2 daysfollowing activation, again in both Th1 and Th2 cultures. However,tertiary activation of T cells revealed selective induction in the Th1cultures, but not in the Th2 cultures (FIG. 13A). These results suggestthat BTLA expression on CD4+ T cells is initially controlled primarilyby T cell activation and not by factors governing Th1 or Th2differentiation. The delayed loss of BTLA inducibility in Th2 cellsmight suggest a silencing process rather than a Th1-specific pathway forinduction, which would be consistent with our initial finding that BTLAexpression is not dependent on Stat4 or Stat1. Finally, the rapidmodulation of BTLA expression, peaking on day 2 and extinguished by day7, suggests that it may act in the mid-phases of T cell activationfollowing interactions with APCs.

In contrast to the activation-dependent expression of BTLA seen in CD4+T cells, BTLA expression on B cells was maintained at high levelsthroughout activation by LPS or anti-lgM stimulation (FIG. 13B). Theseresults differ slightly from the reported 3- to 10-fold decrease in BTLAexpression following LPS activation of B cells. Nonetheless, our resultsagree with that report in the finding of high levels of BTLA expressionon B220+ B cells in the periphery, and to some degree, the constitutivenature of its expression.

Selective Induction of BTLA on Anergic T Cells

Previously, a method of anergy induction for naive CD4+ T cells wasdeveloped that involves adoptive transfer of Ag-specific CD4+ T cellsinto recipients expressing Ag on somatic tissues. Specifically, clone6.5 transgenic T cells, reactive to HA peptide 110-120 presented byI-Ad, become anergic when transferred into recipient mice expressing amembrane bound form of HA targeted for expression on lung and prostatetissue. We analyzed BTLA expression following T cell transfer on variousdays after transfer using Affymetrix gene arrays and FACS (FIGS. 14, Aand B). We found that BTLA mRNA was highly induced in these anergic CD4+T cells in this system, compared with CD4+ T cells activated byAg-expressing vaccinia virus (FIG. 14A). At 2 days after transfer, BTLAexpression by T cells undergoing anergy induction was twice the level ofnaive T cells, and significantly higher than activated T cells. Thisinduction was more evident by day 3 and day 4 following transfer, withBTLA expression about 3-fold higher than in naive T cells. By contrast,BTLA levels were substantially reduced in fully activated T cellscompared with naive or anergic T cells at these times (FIG. 14A). As acontrol, myosin Vlla, a constitutive “housekeeping” gene, showedessentially no change in these three conditions over these times. Thus,BTLA mRNA appears to decline more rapidly than BTLA surface protein inactivated T cells because activated T cells express peak BTLA surfacelevels at day 2 (FIG. 13), but show reduced BTLA mRNA (FIG. 14B). Theseobservations are consistent with the reduced BTLA surface expression byday 4 and the essentially undetectable BTLA expression by day 7.

We next measured BTLA expression by FACS under conditions of anergyinduction or activation (FIG. 14B). Notably, the highest levels of BTLAsurface expression coincided with induction of anergy in vivo.Specifically, 6 days after transfer, anergic T cells expressed about10-fold higher BTLA than naive T cells, and about 3-fold higher than invivo-activated T cells (FIG. 14B). We verified that the CD4+ T cellstransferred into HA-expressing recipients did become anergic as definedby lack of proliferation (FIG. 14C), consistent with previous reports.For comparison, we also wished to evaluate BTLA expression onconventional naive CD4+ T cells (CD4+ CD25−) T cells or T regulatorycells (CD4+ CD25+) either as resting cells ex vivo or after in vitroactivation with anti-CD3 (FIG. 14D). As expected, BTLA was expressed atlow levels on naive T cells, and was induced about 10-fold 36 h afteranti-CD3 treatment. Freshly isolated T regulatory cells expressedsimilar levels of BTLA as freshly isolated naive CD4+ T cell, but showedonly a slight increase after treatment with anti-CD3 (FIG. 14D). As acontrol, we confirmed that T regulatory cells, but not naive T cells,expressed PD-1, consistent with previous reports. As a further control,we showed that the isolated CD25+ T regulatory cells failed toproliferate in vitro, in contrast to the robust proliferation of freshlyisolated naive T cells (FIG. 14E). In summary, BTLA shows a pattern ofexpression that is somewhat distinct from that of CTLA-4 and PD-1 interms of its response to anergy induction and expression by T regulatorycells.

Role of BTLA in T Cell-Independent Ab Responses

Our initial analysis of BTLA was motivated by consideration of its rolein T cell activation. However, the fact that B cells express the highestlevel of BTLA, and the constitutive nature of this expression, motivateda second examination of its effect on Ab production. In our study, weexamined T cell-independent Ab responses using immunization withNP-Ficoll in wild-type mice or BTLA−/−129SvEv mice, which express theBALB/c allele of BTLA. We immunized cohorts of mice with one injectionof NP-Ficoll in alum and measured production of anti-NP Abs of specificisotypes on day 14 (FIG. 15). For the isotypes IgM, IgG1, IgA, we foundno specific changes in levels of anti-NP Abs. For IgG2a or IgG2b, wefound only slight increases in anti-NP Abs in the BTLA−/− compared withwild-type mice. However, for Abs of the IgG3 isotype, which is primarilyassociated with T-independent responses, we found an about 2-foldincreased in anti-NP-specific Abs in BTLA−/− mice compared withwild-type mice. The size of this difference is consistent with therelatively modest increases in B cell and T cell proliferation responsesdescribed for BTLA−/− cells previously, and is consistent with aninhibitory rather than activating role of BTLA. However, the relativelymodest magnitude of this effect could also be an indication that BTLAexpression by B cells may serve a purpose other than cell-intrinsicsignaling, such as perhaps delivery of a signal toward cells expressingligands for BTLA.

Methods (II)

The following Abs used for FACS analysis were from BD Pharmingen:CD4-CyChrome (RM4-5), CD8-FITC (53-6.7), B220-allophycocyanin (RA3-6B2),CDHb-FiTC (M1/70), CD11c-FITC (HL3), DX5-FITC, 1-Ad-PE (AMS-32.1),I-Ab-PE (AF6-120.1), IgM-PerCP Cy5.5 (R6.60.2), CD21/CD25-FITC (7G6),CD25-allophycocyanin (PC61), CD62 ligand-FITC (MEL-14), Thy1.1-PerCP(OX-7), goat anti-mouse Ig-PE, mouse anti-Armenian/Syrian hamster IgG-PE(mixture), Streptavidin (SA)-PE, SA-CyChrome, and SA-aliophycocyanin.KJ1-26 Tricolor, hamster IgG-biotin, and murine IgG1-biotin were fromCaltag Laboratories. All FACS analysis included an initial incubationwith 2.4G2 (anti-CD16/CD32; BD Pharmingen) to block Fc receptorinteractions.

Sequencing of BTLA and PD-1 Ig Domains

Exon 2 of BTLA or PD-1, encompassing the Ig domain, was amplified by PCRfrom genomic DNA from a panel of mouse strains using Easy-A HighFidelity PCR Cloning Enzyme (Stratagene) and the following intronicprimers: BTLA (sense) ATGGTCCTTCTAAGAGTGAAC (SEQ ID NO: 8), (antisense)ATAGATGGTCTGGGGTAGATC (SEQ ID NO:9) and PD-1 (sense) CAGGCTCCTTCCTCACAGC(SEQ ID NO:10), (antisense) CTAAGAGGTCTCTGGGCAG-3′ (SEQ ID NO:11).

PCR products were cloned into the pGEM-T Easy vector (Promega) andinserts from at least three individual subclones from each strain weresequenced using the T7 universal primer.

Generation of Soluble BTLA Ig Domain

The Ig domain of C57BL/6 BTLA was PCR amplified from cDNA using thefollowing primers: BTLA (sense) CATGCCATGGAGAAAGCTACTAAGAGGAAT (SEQ IDNO:12) and BTLA (antisense) CGGGATCCTGAAGAGTTTTGAGTCCTTTC-3′ (SEQ IDNO:13). The product was subcloned into the pET28 vector (Novagen) thathad been modified to contain a BirA biotinylation sequence(GGGLNDIFEAQKI EWHE) (SEQ ID NO:14) onto the C terminus of the BTLA Igdomain. Proteins were expressed as insoluble inclusion bodies in BL21(DE3) Codon Plus RIL cells (Stratagene) and refolded.

Production of mAbs to BTLA

Armenian hamsters or BALB/c background BTLA−/− mice were immunized with100 μg of refolded C57BL/6 BTLA Ig domain protein in CFA, boostedbiweekly with 100 μg of protein in IFA, and received a final i.v. boost3 days before fusion. Splenocytes were fused with the P3X63Ag8 myeloma,and hybridoma supernatants screened for binding to BJAB cells expressingeither C57BL/6 or BALB/c BTLA Ig domains as GFP fusion proteins. TheBTLA-GFP chimera was prepared by splicing by overlap extension (SOEing).A PCR fragment containing the BTLA cDNA with a 3′ tail annealing to the5′ end of GFP was amplified by PCR made using Vent polymerase, theprimers J10RV1-BgIII (AGCTCTGAAGATCTCTAGGGAGGAAG) (SEQ ID NO: 15) and 3′J10+10 (CCTTGCTCACACTTCTCACACAAATGGATGC) (SEQ ID NO: 16) with DO11.10BTLA cDNA as template. A second fragment containing GFP cDNA, withoutits start codon, with a 5′ tail annealing to the 3′ end of BTLA wasamplified by PCR using Vent polymerase and the primers 5′ GFP+10(TGTGAGAAGTGTGAGCAAGGGCGAGGAGC) (SEQ ID NO: 17) and 3′ GFP+Sal(ACGCGTCGACTTACTTGTACAGCTCGTCCATG) (SEQ ID NO: 18) with the GFP cDNA astemplate. The chimeric BTLA-GFP fusion cDNA was amplified by PCR from amixture of these two PCR fragments using the primers J10RV1-BglII and 3′GFP+Sal, digested with BglII and Sail, and cloned into the BglII/SalIsites of IRES-GFP-RV to produce DO11.10-BTLA-GFP-RV. A cytoplasmicdeletion was made using site directed mutagenesis (Stratagene) and theprimers mj11trunc top (GTTGATATTCCAGTGAGCAAGGGCGAGGAG) (SEQ ID NO: 19)and mj11trunc bottom (CTTGCTCACTGGAATATCAACCAGGTTAGTG) (SEQ ID NO: 20)to produce DO11.10-BTLA-trunc-GFP-RV. The C56BL/6 version of BTLAtrunc-GFP-RV was made by purifying a natural Bglil/BamHI fragment from aBTLA cDNA cloned from a mouse spleen cDNA phage library (Stratagene).This fragment was then cloned into the BgIII/BamHI digested DO11.10-BTLAtrunc-GFP-RV to produce C57BL/6-BTLA trunc-GFP-RV.

Positive hybridomas were expanded and Abs purified using MAPS II-proteinA columns. Hamster monoclonal 6A6 is of the IgG isotype, whereas allmurine Abs are IgG1κ. Unless otherwise stated, all Abs were biotinylatedusing EZ-Link Sulfo-NHS-LC-biotin (Pierce) and detected withSA-conjugated fluorochromes. This procedure eliminated secondary Abcross-reactivity with murine cells.

Yeast Display Mapping

The Ig domain of the C57BL/6 BTLA allele was amplified from cDNA usingthe primers 5′-GGAATTCCATATGCAGCCAAGTCCTGCCTG-3′ (SEQ ID NO: 21) and5′-CATGCTAGCGAGAAAGCTACTAAGAGGAA-3′ (SEQ ID NO: 22) and subcloned intothe NdeI and the NheI sites of the pCT302-AGA2d vector to create anHA-tagged fusion to the Aga2 peptide. QuickChange mutagenesis was usedto introduce mutations into this construct using the following primerpairs: C26At,

C26At, (SEQ ID NO: 23) CAGTGCAACTTAATATTACGAGGAATTCCAAACAG; C26Ab,(SEQ ID NO: 24) CTCGTAATATTAAGTTGCACTGGACACTCTT; C32At, (SEQ ID NO: 25)GCAACTTACTATTAAGAGGAATTCCAAACAGTCTGC; C32Ab, (SEQ ID NO: 26)AATTCCTCTTAATAGTAAGTTGCACTGGACA; G48Ct, (SEQ ID NO: 27)GAATCCCAAACACTCTGCCAGGACAGGAGAGT; G48Cb, (SEQ ID NO: 28)CTGGCAGAGTGTTTGGAATTCCTCGTAATAG; A55Tt, (SEQ ID NO: 29)ACAGTCTGCCTGGACAGGAGAGTTATTTAAAATT; A55Tb, (SEQ ID NO: 30)TCCTGTCCAGGCAGACTGTTTTGAATTCCT; C79Gt, (SEQ ID NO: 31)GAGTTATTTAAAATTGAATGTCCTGTGAAATACTGTGT; C79Gb, (SEQ ID NO: 32)AGGACATTCAATTTTAAATAACTCTCCTGTCC; T147Gt, (SEQ ID NO: 33)ATGGAACAATCTGGGTACCCCTTGAGGTTAGCC; T147Gb, (SEQ ID NO: 34)GGGTACCCAGATTGTTCCATTGTGCTTAC; A163G/T168Gt, (SEQ ID NO: 35)TTGAGGTTGGCCCGCAGCTATACACTAG; A163/T168Gb, (SEQ ID NO: 36)GCTGCGGGCCAACCTCAAGGGGTACACAGA; A197Gt, (SEQ ID NO: 37)TTGGGAAGAAAATCGATCAGTTCCGGTTTTTGTTCT; A197Gb, (SEQ ID NO: 38)AACTGATCGATTTTCTTCCCAACTAGTGTA; C320Gt, (SEQ ID NO: 39)ATCCATGTGAGAGAAAGGACTCAAAACTCTTCA; and C320Gb, (SEQ ID NO: 40)AGTCCTTTCTCTCACATGGATGGTTACTGAATG.

Transformation of EBY100.Aga1 yeast with each construct resulted insurface expression of the BTLA mutant. Expression level was confirmed byanti-HA staining. Yeast cells were stained with anti-BTLA Abs asindicated to determine mutations that abolished Ab recognition.

CD4+ T Cell Activation and Expression Analysis

DO11.10 TCR transgenic cells were activated with 0.3 μM OVA peptide(amino acids 323-339) and irradiated (2000 rad) BALB/c splenic APCs. Th1conditions consisted of heat-killed Listeria monocytogenes, IL-2 (40U/ml; Takeda Chemical Industries), and 10 μg/ml anti-IL-4 (11 B11). Th2cells were differentiated in 100 U/ml IL-4, 3 μg/ml anti-IL-12 (TOSH),and IL-2. Cells were restimulated with Ag and APCs on days 7 and 14.Th1/Th2 phenotypes were confirmed at days 7 and 14 by intracellularcytokine staining for IFN-γ and IL-4.

Gene Microarray

Anergic T cells were isolated by adoptively transferring 2.5×10⁶Thy1.1+HA-specific T cells to recipient mice (C3-HAhigh). After 4 daysin vivo, animals were sacrificed via CO₂ asphyxiation. Spleens wereharvested, and subjected to ACK lysis. Adoptively transferredHA-specific T cells were enriched by binding the resulting cells withAbs to CD8a (53-6.7), B220 (RA3-6B2) and Thy1.2 (30-H12), followed byincubation with SA-conjugated magnetic microbeads (Miltenyi Biotec).Unwanted cells were depleted by passage over LS columns (MiltenyiBiotec) according to the manufacturer's protocol. The remaining cellswere stained with an Ab to Thy1.1 (OX-7) and further enriched usingfluorescence-based cell sorting on a FACSVantage TurboSort (BDBiosciences). The resulting populations were between 95 and 99% pure.Cells were kept at 4° C. throughout the enrichment procedure. In vitroassays confirmed the anergic phenotype of the sorted cells. All Abs werepurchased from BD Pharmingen. This procedure specifically avoidsligation of the TCR or CD4 during the isolation process. Activated,memory and naive clonal T cells were isolated in an analogous manner,using a specific viral construct (vaccinia-HA) to activate the cellsafter adoptive transfer to nontransgenic B10.d2 mice. RNA was isolatedfrom each T cell population using the RNAeasy kit according to themanufacturer's instructions (Qiagen), and cRNA probe was prepared.Fragmented cRNA was hybridized to mouse GeneChips MU174A, MU174B, andMU174C per Affymetrix standard hybridization protocol. Each chipcontained about 12,000 different genes (chip A) per expressed sequencetag (EST) with (chips B and C), for a total of about 36,000 genes perEST from the three chips. A single gene/EST was represented by a probeset defined by 16-20 perfect match oligonucleotides that span the lengthof the gene, as well as 16 oligonucleotides with 1 by mismatch. Theintensity of a gene was determined by evaluating the perfect match andmismatch intensities, as described in Affymetrix Microarray Suite,version 5.1 software (Affymetrix). The experiment was replicated once,for a total of two replicate intensities within each condition. Toidentify probe sets associated with an anergic phenotype, we used thehypothesis-based analysis of microarrays algorithm previously described(28) with the boolean hypothesis day 4 anergy>naive AND day 4 anergy>day4 activation.

Assessment of Anergy by Proliferation

On indicated days following transfer of HA-TCR transgenic T cells,20×10⁶ splenocytes were incubated with increasing doses of HA peptide.Proliferation was assayed after 48 h, with a [³H]thymidine pulse in thefinal 12 h.

BTLA Expression by Naive, Activated, and Anergic CD4+ T Cells

HA-TCR transgenic T cells were enriched by depletion of CD8+ and B220+cells as earlier described. Cells were CFSE-labeled as previouslydescribed (29) before adoptive transfer of 2.5×10⁶ clonotypic cells viatail vein injection. Cells were stained with anti-Thy1.1 PerCP and theanti-BTLA Ab 6F7-biotin, followed by SA-PE.

Purification and Activation of CD4+ CD25+T Regulatory Cells

Splenocytes and lymph node cells from BALB/c mice were isolated.Following erythrocyte lysis, B220+ cells were depleted by magneticseparation with anti-B220 Microbeads (Miltenyi Biotec). The negativefraction was stained with CD25-PE (BD Pharmingen) and anti-PE Microbeads(Miltenyi Biotec) and magnetically separated into CD25+ andCD25-fractions. Enrichment was assessed by FACS as shown (see FIG. 14D).Contaminating non-CD4+ cells were mainly B220+ or CD8+ cells. Toactivate T cells, 1×10⁶ cells/ml of each fraction were cultured onflat-bottom plates coated with 10 μg/ml 2G11 (anti-CD3; BD Pharmingen)for 48 h. Cells were pulse with 1 μCi/well [³H]thymidine for anadditional 12 h.

Ab Response to NP-Ficoll

Eight-week-old BTLA+/+ and BTLA−/− littermate mice on the 129SvEvbackground were immunized i.p. with 50 μg of nitrophenyl (NP)-Ficoll(Biosearch Technologies) in Imject alum (Pierce). Sera were collected onday 14, and the titers of anti-NP were determined by ELISA usingNP25-BSA (Biosearch Technologies) for Ab capture and the SouthernBiotechnology clonotyping/HRP kit for IgG subclass-specific ELISA(Southern Biotechnology Associates).

For further details regarding Example II, including references, seeHurchla et al., J. Immunol., 174: 3377-3385, 2005, which is expresslyincorporated herein in its entirety by reference.

(III) BTLA-HVEM Effects in Graft Survival

BTLA and HVEM Regulate Acceptance of Partially MHC-Mismatched CardiacAllografts

Primarily vascularized cardiac allografts are the most frequent organtransplant undertaken in mice and may be performed across full MHCdisparities, with rejection in 7-8 days, or across MHC class I or IIdisparities, which leads to long-term survival (>100 days). The basisfor this unexpectedly long-term survival of cardiac transplants acrosspartial MHC disparities is unknown and has received little attention. Asanticipated from the literature, we indeed found that cardiac allograftsperformed across an MHC class II mismatch (Bm12 B6) survived long termin wild-type recipients (mean survival time (MST), >100 days; n=6).Histologic assessment of these allografts harvested at 2 wk aftertransplant showed preservation of myocardial architecture and generallyonly sparse mononuclear cell infiltration (FIG. 29 a). In contrast,BTLA−/− recipients rejected Bm 12 cardiac allografts by 2-3 wk aftertransplant (MST, 14.3±3.8 days; n=12; p<0.001), and histology showed amarked increase in leukocyte infiltration and myocardial injury (FIG. 29a). In addition, comparable abrogation of Bm 12 allograft survival wasseen with mAb targeting of BTLA in wild-type recipients (MST, 23.2±3.2;n=6; p<0.001) or by engraftment of recipients lacking the BTLA ligand,HVEM (MST, 17.4±4.2 days; n=8; p<0.001; FIG. 29 a). Thus, BTLA and HVEMare required to allow long-term survival of partially mismatched cardiacallografts. In contrast to results obtained with BTLA−/− recipients,PD-1−/− recipients receiving Bm12 cardiac allografts exhibited an 80%long-term allograft survival (FIG. 29 b), although we did observe aminor role for PD-1 in regulating responses to Bm12 cardiac allografts.Dual BTLA−/− and PD-1−/− knockout mice (DKO) mice rejected Bm12 donorhearts more rapidly (MST, 10.5+1.5 days; n=4) than singly deficientBTLA−/− recipients (p<0.05) or wild-type controls (p<0.0001; FIG. 29 b).

Like MHC class 11-mismatched grafts, MHC class 1-mismatched (Bm1 B6)cardiac allografts survived long term when transplanted to wild-type B6mice, but were rejected in BTLA−/− mice (FIG. 29 c). Furthermore, incontrast to wild-type B6 recipients, the MHC class 1-mismatchedallografts in BTLA−/− recipients showed increased mononuclear cellinfiltration and progressive tissue damage indicative of the developmentof cellular rejection (FIG. 29 c). PD-1−/− recipients receiving Bm1cardiac allografts had 100% long-term allograft survival. Collectively,these findings indicate that BTLA, in contrast to PD-1, is capable ofinhibiting the generation of a functional allogeneic immune response inthe context of partial MHC mismatches.

BTLA Suppresses MHC Class II-Dependent T Cell Responses

The unexpected rejection of Bm12 allografts by BTLA−/−, but not PD-1−/−,mice suggested that BTLA and PD-1, or their ligands, might bedifferentially expressed in partially MHC-mismatched allografts. BTLAmRNA expression within Bm12 allografts was 20-fold higher than PD-1 at 7days after transplant, whereas no BTLA expression was detected within Bm12 hearts engrafted into BTLA-−-recipients, indicating BTLA expressionprimarily by infiltrating host leukocytes (FIG. 30 a). Comparable BTLAexpression was observed within long-surviving allografts (data notshown). Unlike BTLA, only very low levels of PD-1 were detected in Bm 12allografts in either wild-type or BTLA−/− recipients (FIG. 30 a). Nodifferences in the levels of expression of HVEM, PD-L1, or PD-L2 wereseen between wild-type and BTLA−/− recipients (FIG. 30 a). These datasuggest that in the Bm12 B6 model, BTLA is the predominant inhibitoryreceptor expressed by infiltrating alloreactive T cells, and that in theabsence BTLA, there is no compensatory increase in expression ofadditional inhibitory molecules.

We next studied the in vitro and in vivo responses of T cells fromwild-type and BTLA−/− mice to MHC class II Ags. First, we examined thein vitro proliferation of purified wild-type or BTLA−/− CD4+ T cellscocultured with irradiated Bm12 DC. Proliferation of BTLA−/− T cells wasincreased compared with that of wild-type T cells, as measured by eitherBrdU incorporation (FIG. 30 b) or CFSE dilution (FIG. 30 c). To assessin vivo responses, 40 million CFSE-labeled wild-type or BTLA−/−splenocytes were adoptively transferred into irradiated Bm 12 hosts, anddonor CD4+ T cell proliferation was assessed. Although a large portionof wild-type CD4+ T cells remained undivided 72 h after adoptivetransfer, almost all BTLA−/− CD4+ T cells had entered the cycle andproceeded through several rounds of division (FIG. 30 d). Hence, BTLAregulates CD4+ T cell alloactivation and proliferative responses to MHCclass II Ags.

MHC class II-restricted CD4+ T cell proliferation dominates hostalloresponses in the Bm12 B6 model, although host responses are known toinclude stimulation of CD8+ precursor CTL by class II-restricted CD4+ Tcells. We found that although proliferative responses of CD8+ T cells inirradiated Bm 12 hosts were low compared with those of CD4 cells, thealloactivation and proliferation of CD8+ T cells from BTLA−/− mice weremarginally increased over control cells in this assay system (FIG. 30e). We examined recipient anti-donor responder frequencies by ELISPOT,with the readout of IFN− spot-forming cells by recipient splenocytes.BTLA−/− recipient splenocytes had significantly higher anti-donorresponder frequencies when challenged with Bm12 APCs (FIG. 30 f),consistent with the increased allogeneic proliferation in vitro and theaccelerated graft rejection in vivo of T cells from BTLA−/− mice.

Minor Role of BTLA in Fully MHC-Mismatched Alloresponses

We next tested whether BTLA played a similar dominant role in regulatingresponses to fully MHC-mismatched cardiac allografts as it did forpartially MHC-mismatched cardiac allografts. Wild-type recipients (B6,H-2b) rejected cardiac grafts (BALB/c, H-2d) in 7-10 days (MST, 8±1days; n=6), whereas BTLA−/− recipients showed a small and unexpectedprolongation of graft survival (MST, 12±5 days; n=6; p<0.05; FIG. 31 a).In addition, wild-type mice treated with a neutralizing anti-BTLA mAbshowed a similar prolongation of allograft survival (MST, 13±1 days;n=4; p<0.05) compared with control IgG treated recipients (MST, 8+1days; n=4; FIG. 31 b). Furthermore, addition of a subtherapeutic courseof rapamycin prolonged graft survival in wild-type mice by a few days(MST, 11±2 days; n=6; p<0.05), but significantly prolonged graftsurvival in BTLA−/− mice (MST, 53±12 days; n=8; p<0.001), with 25% ofthe latter recipients achieving long-term acceptance (FIG. 31 c). Hence,in the case of fully MHC-mismatched cardiac allografts, loss of BTLA didnot accelerate allograft rejection, but, rather, caused a surprising,albeit small, increase in allograft survival. By contrast, the presenceor the absence of BTLA had no effect on the tempo of rejection of B6cardiac allografts by BALB/c recipients; all allografts were rejectedwithin 7-10 days (n=4/group; p>0.05).

To understand the prolongation of fully MHC-mismatched graft survival,we measured the expression of cytokines and chemokine receptorsimportant to host T cell recruitment in this model), using allograftsharvested 7 days after transplant. We found decreases in IL-2 and IFN−mRNA in BTLA−/− recipients compared with wild-type recipients (FIG. 31d). We also found reduced expression of CXCR3 and CCR5 in BTLA−/−recipients compared with wild-type recipients (FIG. 31 d). Therapy withrapamycin accentuated differences in cytokine and chemokine receptormRNA expression between BTLA−/− and wild-type recipients (FIG. 31 d).Given a key role for IFN−-induced IFN− inducible protein 10 (IP-10)production in promoting CXCR3+ cell recruitment and allograft rejectionin this model, we performed Western blotting, which confirmed thatallografts in BTLA−/− recipients had reduced IP-10 and CXCR3 proteinscompared with wild-type controls, with or without rapamycin therapy(FIG. 31 e).

To assess whether the lack of BTLA affected the strong alloactivationand proliferation induced in T cells by 72 h in this model, we used theparent-to-F1 model involving transfer of CFSE-labeled cells across fullyallogeneic barriers (FIG. 31 f). In this model, the activation and cellcycle progression of CD4+ responses were similar for BTLA−/− andwild-type cells, and CD8+ T cells from BTLA−/− mice were only marginallydecreased compared with those from wild-type controls (FIG. 31 f).However, the evaluation of intracellular cytokine production byalloreactive T cells showed decreased IL-2 and IFN− production byalloreactive BTLA−/− CD4+ and CD8+ T cells compared with wild-type Tcells (FIG. 31 g). Again, a subtherapeutic rapamycin dose caused amodest decrease in proliferation of BTLA−/− T cells compared withwild-type T cells, particularly CD8+ responses (FIG. 31 f), anddecreased production of IL-2 and IFN− by both T cell subsets (FIG. 31g). These data indicate that T cell activation, proliferation, andproduction of cytokines such as IL-2 and IFN− are decreased in BTLA−/−mice, especially when recipients are treated with limitedimmunosuppression, and that these impaired responses are associated withmodulation of chemokine/chemokine receptor effector pathways.

Involvement of PD-1 and BTLA in Fully MHC-Mismatched Alloresponses

In considering explanations for the differing effects of BTLA in thepartial MHC-mismatch and full MHC mismatch models, we wondered whetherdifferential reliance on PD-1 between these models might play a role.Therefore, we examined the contributions of both PD-1 and BTLA in thefully MHC− mismatched model (FIG. 32). We found, first, that BALB/ccardiac allografts were rejected at similar rates (p>0.05) by C57BL/6wild-type mice and DKO mice (FIG. 32 a). Second, consistent with the DKOdata, mAb blockade of PD-1 increased the rate of rejection of fullyMHC-mismatched allografts by BTLA−/− recipients (p<0.001; FIG. 32 b).Third, the duration of allograft survival in BTLA−/− recipientsreceiving subtherapeutic course of rapamycin (MST, 53±12 days; n=8) wasmarkedly decreased by loss of PD-1, as seen by examining either DKOrecipients (MST1 12.8±2.2 days; n=4; p<0.001; FIG. 32 c) or by mAb Abblockade of PD-1 in BTLA−/− mice (MST, 14.0±3.5 days; n=4; p<0.001; FIG.32 d). In summary, in contrast to partial MHC-mismatched allografts, theresponses against fully MHC-mismatched cardiac allografts are regulatedby both BTLA and PD-1.

We next asked whether PD-1 regulated the proliferation and function of Tcells responding to fully MHC-mismatched allografts. Analysis by qPCR ofBALB/c cardiac allografts harvested on day 7 after transplant fromC57BL/6 recipients showed intragraft expression of BTLA, PD-1, and theirligands, HVEM, PD-L1, and PD-L2 (FIG. 33 a). By comparison withwild-type recipients, BALB/c allografts harvested from BTLA−/−recipients had increased PD-1 expression (FIG. 33 a; p<0.01). Incontrast to PD-1 expression, the expression of HVEM, PD-L1, and PD-L2was not increased in BTLA−/− recipients. These results suggest that inthe absence of BTLA, host leukocytes might express more PD-1 in responseto allostimulation.

To directly examine PD-1 expression by alloreactive wild-type or BTLA−/−T cells, we adoptively transferred CFSE-labeled splenocytes intoirradiated Bm12 (class II-mismatched) or B6D2F1 (fully MHC-mismatched)recipients. At analysis 72 h later, we found that in the MHC class IIpartial mismatch, PD-1 was weakly expressed by alloreactive CD4+ Tcells, but not at all by CD8+ T cells, from wild-type or BTLA−/− mice(FIG. 33 b). In contrast, with a full MHC mismatch, PD-1 expression byboth CD4+ and CD8+ donor T cells was markedly increased, and the extentof PD-1 expression was higher in BTLA−/− vs wild-type T cells (X FIG. 33b). Moreover, treatment with rapamycin reduced PD-1 expression bywild-type T cells, but had only minor effects on PD-1 induction by Tcells from BTLA−/− mice (FIG. 33 b).

Lastly, we used in vivo and in vitro approaches to examine the roles ofBTLA and PD-1 in regulating T cell proliferation and cytokine productionin response to fully MHC-mismatched allostimulation (FIG. 33, c-e).Compared with wild-type or BTLA−/− cells, DKO cells showed enhancedproliferation (FIG. 33 c) and Th1 cytokine production (FIG. 33 d).Therapy with rapamycin decreased the alloactivation-inducedproliferation (FIG. 33 c) and cytokine production (FIG. 33 e) of CD4+and CD8+ T cells from wild-type and BTLA−/− donors, but did not blockthese events in DKO CD4+ or CD8+ T cells (FIGS. 33, c and e). Indeed,the production of IL-2 and IFN− was increased in DKO T cells comparedwith wild-type and BTLA−/− T cells (FIG. 33 d), including in thepresence of rapamycin therapy (FIG. 33 e). Collectively, these dataindicate that 1) PD-1 expression is highly induced on the surfaces ofalloreactive CD4+ and CD8+ T cells upon exposure to fully MHC-disparateallografts; 2) the levels of PD-1 on alloreactive CD4+ and CD8+ T cellsare still further increased in the absence of BTLA; and 3) increasedPD-1 expression is associated with inhibitory effects on thealloantigen-induced production of cytokines such as IL-2 and IFN−. Inassociated in vitro studies, as T cell activation increased in responseto allogeneic DC (FIG. 34), the induction of PD-1 was increasinglyapparent compared with that of BTLA. BTLA up-regulation occurred upon Tcell activation, but did not show expansion comparable with that of PD-1with increasing T cell activation, suggesting that the strength of Tcell activation determines the relative importance of these twopathways.

Methods (III)

Mice

BTLA−/−, PD-1−/−, and dual BTLA−/− and PD-1−/− mice were backcrossed formore than eight generations on a C57BL/6 background; HVEM−/− mice weregenerated by homologous recombination and backcrossed more than fivegenerations on a B6 background. Wild-type C57BL/6 (H-2b), BALB/c (H-2d),C57BL6/DBA F1 (H-2b/d), Bm12 (B6.C-H2bm12/KhEg), and Bm1(B6.C-H2bm1/ByJ) mice were purchased from The Jackson Laboratory, housedin specific pathogen-free conditions, and used for studies approved bythe institutional animal care and use committee of Children's Hospitalof Philadelphia.

An Armenian hamster anti-mBTLA neutralizing mAb, 6A6, was describedpreviously in Sedy et al. Nature Immunol. 6:90-98 (2005), and wepurchased mAbs for flow cytometry (BD Pharmingen) and Abs for Westernblotting (Santa Cruz Biotechnology). Labeling of cells with CFSE(Molecular Probes) was undertaken as previously reported.

Quantitative PCR (qPCR)

We performed qPCR as previously described. Briefly, RNA was extractedwith TRIzol (Invitrogen Life Technologies), RT of random hexamers wasperformed with an ABI PRISM 5700 unit (Applied Biosystems), and specificprimer and probe sequences for target genes were used for qPCRamplification of total cDNA (TaqMan PDAR; Applied Biosystems). Relativequantitation of target cDNA was determined using a control value of 1;the sample cDNA content was expressed as the fold change from thecontrol value. Differences in cDNA input were corrected by normalizingsignals obtained with specific primers to ribosomal RNA; nonspecificamplification was excluded by performing RT-PCRs without target cDNA.

Flow Cytometry

Alloreactive T cells were generated by i.v. injection of 40×10⁶CFSE-labeled B6 spleen and lymph node cells into B6/DBA F1 recipients, aparent F1 MHC mismatch in which only donor cells respond. Splenocytesharvested after 3 days were incubated with CD4-PE, CD8-PE, CD25-PE,CD44-PE, CD62L-PE, PD-1-PE, ICOS-PE, and biotin-conjugated anti-H-2 Kdand anti-H-2Dd mAb. Donor alloreactive T cells were identified by gatingon H-2 Kd and H-2Dd cells (FACSCalibur; BD Biosciences), and theirproliferation was assessed by CFSE division profiles. For intracellularcytokine staining, splenocytes (3×10⁶/ml) were treated with Golgi-Stop(BD Pharmingen), stimulated for 4 h with PMA (3 ng/ml) and ionomycin (1μM) in 24-well plates in complete medium (RPMI 1640, 10% FCS, 100 U/mlpenicillin, 100 μg/ml streptomycin, and 50 μM 2-ME), and stained withcell surface markers (CD4-PE or CD8-PE, biotin-conjugated H-2 Kd orH-2Dd, followed by streptavidin-PerCP), fixed, and stained withIFN−-allophycocyanin or IL-2-allophycocyanin after permeabilization(Perm-Wash buffer; BD Pharmingen).

In Vitro Cellular Assays

For propagation of bone marrow-derived DC, bone marrow cells harvestedfrom the femurs and tibia were cultured for 5-7 days in 24-well plates(2×10⁶/well) in medium plus mouse GM-CSF (5 ng/ml) and IL-4 (10 ng/ml)(20, 21). One-way MLR cultures were performed in triplicate, usingmagnetic column-eluted splenic T cells (2×10⁵/well) as responders andgamma-irradiated (20 Gy) DC as stimulators. Cultures were maintained incomplete medium for 72-96 h, and T cell proliferation was determined byBrdU incorporation or CFSE dilution profile. BrdU staining with a BrdUlabeling kit (BD Pharmingen) was performed using the manufacturer'sinstructions. Cells were pulsed with BrdU, treated with FcR-blockingCD16/CD32 mAbs, stained with cell surface markers, fixed, permeabilized,treated with DNase/Triton X-100, stained with anti-BrdII mAb, andanalyzed by flow cytometry.

ELISPOT

Immunospot assays for IFN− were performed by coating ELISPOT plates (BDPharmingen) with anti-IFN− mAb, blocking, and addition of respondercells isolated from cardiac transplant recipients plus donor splenocytesor bone marrow-derived DC as stimulators; recipient splenocytes or DCwere used as syngeneic controls. At 24 h, cells were discarded, andwells were washed, followed by biotinylated anti-IFN− mAb,streptavidin-HRP, and substrate. Spots were counted using an ImmunospotAnalyzer (Cellular Technology), and recipient anti-donor responderfrequency was determined as the number of IFN− spot-forming cells per10⁶ splenocytes.

Western Blots

Grafts were sonicated in lysis buffer containing Triton X-100 andprotease inhibitors, followed by centrifugation and assay of supernatantprotein content. Proteins were reduced, separated by SDS-PAGE, andtransferred to nitrocellulose membranes. Membranes were blocked,incubated with primary and HRP-linked secondary Abs, and, after thesubstrate reaction, analyzed using National Institutes of Health Image.

Transplantation

Intra-abdominal vascularized cardiac allografting was performed aspreviously described using 6- to 8-wk-old mice. Briefly, donor ascendingaorta and pulmonary artery were anastomosed end-to-side to recipientinfrarenal aorta and inferior vena cava, respectively. Graft survivalwas assessed twice daily by abdominal palpation; rejection was definedas total cessation of cardiac contraction and was confirmed byhistology.

Immunopathology

Portions of harvested allografts were fixed in formalin,paraffin-embedded or snap-frozen, and analyzed by immunoperoxidasestaining with mAbs and an Envision kit (DakoCytomation).

Statistics

Allograft survival was used to generate Kaplan-Meier survival curves,and comparison between groups was performed by log-rank analysis.

For further details regarding Example III, including references, see Taoet al., J. Immunol., 175:5774-5782, 2005, which is expresslyincorporated herein in its entirety by reference.

(IV) BTLA-HVEM Effects in Asthma

Regulated Expression of PD-1 and BTLA During Acute Allergic AirwayInflammation.

We first determined the kinetics of lymphocyte accumulation and receptorexpression in vivo by examining the cells recovered in thebronchoalveolar lavage (BAL) fluid. Mice were systemically sensitizedand challenged with OVA. At 1, 3, 4, or 7 days following challengegroups of mice were euthanized and BAL performed. On 1 day followingchallenge, few CD4+ T cells were found in the BAL fluid. Significantlyincreased numbers of CD4 T cells appeared by day 3 which peaked by day 7post-challenge (FIG. 25). We next examined the expression of PD-1 andBTLA on CD4 T cells recovered in the BAL fluid. Consistent with previousreports that PD-1 expression is induced on activated cells, we foundthat PD-1 expression gradually increased, being detectable on day 3 andreaching its maximum on day 7 following challenge. BTLA expressionexhibited a reciprocal pattern with expression being greatest on day 3and nearly undetectable by day 7 (FIG. 25).

Given the distinct patterns of expression of these receptors on BAL Tcells, we next examined the phenotype of mice deficient either BTLA orPD-1 in the acute allergic airway inflammation model (FIG. 26). BothBTLA-deficient and PD-1-deficient mice showed some increase ininflammatory cell recruitment compared to wild type mice (FIGS. 26 a and26 b). All genotypes had a mixed inflammatory cell infiltrate, althoughthere was an increased percentage of neutrophils and eosinophils in theBTLA-deficient mice (FIGS. 26 a and 26 b). Examination of the lungtissues revealed a mild increase in the intensity of inflammatoryinfiltrates in PD-1 and BTLA-deficient animals compared to wild typecontrols. Thus, while PD-1 and BTLA have been reported as being potentinhibitory receptors, we found only a relatively mild increase in theinflammatory response following of acute allergic airway inflammation inthe absence of either of these inhibitory receptors.

Delayed expression of ligands for BTLA and PD-1 in acute allergic airwayinflammation.

Given the documented inhibitory activity of BTLA and PD-1 in vivo, wewere surprised that the absence of either of these receptors did nothave a greater effect on acute airway inflammation. We thereforespeculated that the ligands for these receptors might not be expressedthereby not allowing this axis of regulation to be apparent. We examinedthe expression of mRNA for Herpes Virus Entry Mediator (HVEM), theligand for BTLA, and PD-L1 and PD-L2, the ligands for PD-1, during anextended time course of airway inflammation (FIG. 27). Expression ofHVEM message was nearly undetectable in the first four days of acuteallergic airway inflammation following challenge but became apparent byday 7 and was maximal by day 10 and 15 (FIG. 25, upper panels).Likewise, the expression of PD-L1 was first detectable at day 2, butremained relatively low in expression until approximately day 7 to day10. Expression of PD-L2, a second ligand for PD-1, was maximum at day 4following intranasal challenge, and declined subsequently.Interestingly, both HVEM and PDL1 were detectable in RNA samplesobtained from cultured murine tracheal epithelial cells (mTEC),suggesting that the source of ligand may be non-immune cells of thelung.

BTLA and PD-1 Limit the Duration of Acute Allergic Airway Inflammation.

Because the ligands for PD-1 and BTLA were maximally expressed in thesecond week following intranasal challenge, we next examined BAL cellnumbers and compositions at day 10 and day 15 following intranasalchallenge (FIG. 28). Wild type mice had completely resolved theinflammation, as evidenced by a low number of cells recovered in the BALfluid and histology at days 10 and 15 following challenge. In starkcontrast, mice deficient in BTLA and PD-1 showed a persistent increasein BAL cells on day 10 following intranasal challenge. Furthermore, thecomposition of these cells in this fluid revealed a greater proportionof lymphocytes and eosinophils in comparison to the few cells in thewild type mice, which consisted predominantly of macrophages. Even onday 15, examination of BTLA-deficient mice revealed the continuedpresence increased numbers of lymphocytes and eosinophils. Directhistological examination of H and E stained sections also demonstratedpersistent inflammation in the lungs of both PD-1 and BTLA-deficientmice at days 10 and 15, whereas the wild type mice had completeresolution in this time frame. Thus, these receptors are critical forthe normal resolution of airway inflammation.

T cell dependent immune responses are determined by the coordinateintegration of signals derived from both cell:cell interactions andsoluble mediators. We have recently described a novel role for CD28signaling not only in the early priming phase but also in maintenance ofthe effector phase of allergic airway inflammation. These studiesfocused on an acute model, acting between days 1 and 3. By contrast, thepresent results show that the inhibitory receptors BTLA and PD-1 exert aslight effect in attenuating the degree of acute inflammation but have aprofound effect on the duration of inflammation, suggesting they act toterminate the immune response. We also observed a temporal regulation ofexpression of the ligands for these receptors during the course of theinflammatory response. Therefore, these data support that the regulatedexpression of inhibitory receptors on lymphocytes and their ligands inthe lung are critical for the proper termination of the acuteinflammatory response. In the absence of the inhibitory receptors onlymphocytes, the normally self limited acute inflammatory responseprogresses to a chronic infiltrate that persists for at least 15 days.We propose, based on these findings, that abnormalities in this axiscould play a role in pathologic situations such as chronic persistentasthma and may represent novel targets for therapeutic intervention.

Methods (IV)

Mice

BTLA-deficient mice were generated as previously described. PD-1deficient mice were obtained from Tasuka Honjo (Kyoto University, KyotoJapan). C57BL/6 mice were purchased from Jackson Laboratories (BarHarbor, Me.). All mice were housed in specific pathogen free facilitiesat Washington University School of Medicine. All animal studies havebeen approved by the Washington University Animal Studies Committee.

Antibodies

Anti-BTLA antibody (Clone 6F7, mouse IgG1) was generated as previouslydescribed. Anti-PD-1 and anti-CD4 antibodies were purchased fromEbiosciences. Flow cytometric analysis was performed on a FacsCaliburcytometer using Cellquest software (Becton Dickinson Corporation).Analysis was performed using FIoJo software.

RT-PCR

Total RNA was extracted from lung tissue of control or allergenchallenged mice using Trizol (Invitrogen). Random primed cDNA wasprepared using the Retroscript kit (Ambion). Specific primers for PDL1,PDL2 and HVEM were designed that spanned intronic sequences. Controlprimers amplify ribosomal S15 RNA and are provided with the RetroscriptKit.

Experimental Allergic Airway Inflammation

Mice were sensitized and challenged with Ovalbumin as previouslydescribed (13). Briefly, mice were injected i.p. with Ova adsorbed toalum on days 0 and 7. On day 14 they received an intranasal challenge of50 μl of 1% Ova in the morning and afternoon. Samples were collected aspreviously described on the indicated days following inhaled challenge.

Preparation of murine tracheal epithelial cells: Primary mouse airwayepithelial cells were cultured and differentiated using an establishedhigh fidelity model of the mouse airway. Briefly, epithelial cells wereharvested from mouse tracheas of C57/BI6 strain mice (5-6 weeks old)using pronase digestion. Cells were purified by differential adherenceof fibroblasts to yield a preparation composed of greater than 99%epithelial cells determined by expression of cytokeratin. Mouse trachealepithelial cells (MTEC) were cultured in the presence of growth factorsupplemented media on semi-permeable membranes (Transwell,Corning-Costar, Corning, N.Y.). Media was maintained in upper and lowerchambers until the transmembrane resistance was greater than1,0000-hms-cm2 indicating tight junction formation. Media was thenremoved from the upper chamber to establish an air-liquid interface(ALI) condition used for epithelial cell differentiation. Cells weredifferentiated for at least 7 days at ALI to generate a multilayer modelof the airway composed of ciliated, secretory, and basal airwayepithelial cells. RNA was prepared from day 7 ALI cultures using Trizolreagent.

(V) Effect of BTLA Loss of Function on Humoral Response

We immunized cohorts of mice with one injection of NP-Ficoll in alum andmeasured production of anti-NP antibodies of specific isotypes on day14. For the isotypes IgM, IgG1, IgA, we found no specific changes inlevels of anti-NP antibodies. For IgG2a or IgG2b, we found only slightincreases in anti-NP antibodies in the BTLA−/− compared to wild typemice. However, for antibodies of the IgG3 isotype, which is primarilyassociated with T-independent responses, we found approximately atwo-fold increased in anti-NP specific antibodies in BTLA−/− micecompared to wild type mice.

In addition, spontaneous germinal centers have been observed at a higherfrequency than control in aging BTLA−/− mice.

(VI) BTLA Modulates Response to Viral Infection

Wildtype and BTLA knockout mice were infected with Sendai virus andmonitored for three weeks. BTLA knockout mice maintained higher bodyweight and exhibited greater survival following infection with Sendaivirus. Similar results were obtained using West Nile virus.

1. An antibody that specifically binds to a protein comprising the aminoacid sequence set forth in SEQ ID NO:2, wherein the antibody is capableof reducing or eliminating phosphorylation of intracellular domaintyrosine residues of said protein or binding of said protein to SHP-2,wherein the antibody is capable of reducing binding of said protein toan HVEM protein.
 2. An anti-BTLA antibody capable of reducing oreliminating phosphorylation of BTLA intracellular domain tyrosineresidues or binding of BTLA to SHP-2, wherein the antibody is capable ofreducing binding of the protein comprising the amino acid sequence setforth in SEQ ID NO: 2 to an HVEM protein.
 3. An antibody thatspecifically binds to an epitope in the Ig domain of a BTLA protein, theBTLA protein comprising the amino acid sequence set forth in SEQ IDNO:2, wherein the antibody is capable of reducing or eliminatingphosphorylation of BTLA intracellular domain tyrosine residues orbinding of BTLA to SHP-2, wherein the antibody is capable of reducingbinding of the protein comprising the amino acid sequence set forth inSEQ ID NO: 2 to an HVEM protein.
 4. An antibody that specifically bindsto an epitope of a human BTLA protein, the epitope comprising one ormore residues selected from the group consisting of V36, Q37, L38, L49,E57, C79, K93, and S96 of SEQ ID NO:2, wherein the antibody is capableof reducing or eliminating phosphorylation of BTLA intracellular domaintyrosine residues or binding of BTLA to SHP-2, and wherein the antibodyis capable of reducing binding of the protein comprising the amino acidsequence set forth in SEQ ID NO: 2 said to an HVEM protein.